U.S. patent application number 15/910519 was filed with the patent office on 2018-07-19 for novel cell-penetrating compositions and methods using same.
The applicant listed for this patent is Yale University. Invention is credited to Frank J. GIORDANO, William C. SESSA.
Application Number | 20180201649 15/910519 |
Document ID | / |
Family ID | 53199565 |
Filed Date | 2018-07-19 |
United States Patent
Application |
20180201649 |
Kind Code |
A1 |
SESSA; William C. ; et
al. |
July 19, 2018 |
NOVEL CELL-PENETRATING COMPOSITIONS AND METHODS USING SAME
Abstract
The invention includes an isolated transport peptide, which
crosses the cell membrane of a cell and/or binds to a target cell.
The invention also includes a transport construct in which a
transport peptide is linked to a cargo moiety to be delivered into
a cell. The invention further includes a method of delivering a
transport construct into and/or to a cell.
Inventors: |
SESSA; William C.; (Madison,
CT) ; GIORDANO; Frank J.; (Madison, CT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Yale University |
New Haven |
CT |
US |
|
|
Family ID: |
53199565 |
Appl. No.: |
15/910519 |
Filed: |
March 2, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15036849 |
May 16, 2016 |
9908915 |
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PCT/US2014/066619 |
Nov 20, 2014 |
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15910519 |
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61908963 |
Nov 26, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 14/4702 20130101;
A61K 38/00 20130101; C07K 14/4703 20130101; C07K 7/06 20130101;
C07K 2319/01 20130101; A61K 47/645 20170801 |
International
Class: |
C07K 7/06 20060101
C07K007/06; C07K 14/47 20060101 C07K014/47; A61K 47/64 20170101
A61K047/64 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with government support under
HL064793, HL061371, HL096670 and HL081190 awarded by National
Institutes of Health. The government has certain rights in the
invention.
Claims
1. An isolated transport peptide, or a salt or solvate thereof,
comprising the amino acid sequence RRPPR (SEQ ID NO: 1).
2. The transport peptide of claim 1, which consists of SEQ ID NO:
1.
3. The transport peptide of claim 1, which is part of a
pharmaceutical composition further comprising a pharmaceutically
acceptable carrier.
4. An isolated transport construct, or a salt or solvate thereof,
comprising a transport peptide comprising SEQ ID NO: 1 linked to a
cargo moiety.
5. The transport construct of claim 4, wherein the transport
peptide consists of SEQ ID NO: 1.
6. The transport construct of claim 4, wherein the cargo moiety is
at least one selected from the group consisting of a nucleic acid;
peptide; protein; oligosaccharide; lipid; glycolipid; lipoprotein;
small molecule compound; therapeutic drug; UV-vis, fluorescent or
radioactive label; imaging agent; diagnostic agent; prophylactic
agent; liposome and virus.
7. The transport construct of claim 4, wherein the cargo moiety is
covalently linked to the transport peptide through a linker or a
chemical bond.
8. The transport construct of claim 7, wherein the linker comprises
a disulfide bond or wherein the chemical bond between the cargo
moiety and the transport peptide comprises a disulfide bond.
9. The transport construct of claim 7, wherein the cargo moiety
comprises a peptide moiety, and wherein the transport peptide is
covalently linked through an amide bond to the N-terminus or
C-terminus of the peptide moiety of the cargo moiety.
10. An isolated transport construct, or a salt or solvate thereof,
comprising a transport peptide comprising SEQ ID NO:1 that is
linked to a cargo moiety comprising the sequence selected from the
group consisting of SEQ ID NOs: 3-6.
11. The transport construct of claim 10, wherein the cargo moiety
comprises a peptide moiety, and wherein the transport peptide is
covalently linked through an amide bond to the N-terminus or
C-terminus of the peptide moiety of the cargo moiety.
12. The transport construct of claim 11, which comprises at least
one sequence selected from the group consisting of SEQ ID NO: 1-SEQ
ID NO: 3; SEQ ID NO: 1-SEQ ID NO: 4; SEQ ID NO: 1-SEQ ID NO: 5; SEQ
ID NO: 1-SEQ ID NO: 6; SEQ ID NO: 3-SEQ ID NO: 1; SEQ ID NO: 4-SEQ
ID NO: 1; SEQ ID NO: 5-SEQ ID NO: 1; and SEQ ID NO: 6-SEQ ID NO:
1.
13. The transport construct of claim 11, which is selected from the
group consisting of SEQ ID NO: 1-SEQ ID NO: 3; SEQ ID NO: 1-SEQ ID
NO: 4; SEQ ID NO: 1-SEQ ID NO: 5; SEQ ID NO: 1-SEQ ID NO: 6; SEQ ID
NO: 3-SEQ ID NO: 1; SEQ ID NO: 4-SEQ ID NO: 1; SEQ ID NO: 5-SEQ ID
NO: 1; and SEQ ID NO: 6-SEQ ID NO: 1.
14. The transport construct of claim 10, which is part of a
pharmaceutical composition further comprising a pharmaceutically
acceptable carrier.
15. A composition comprising an isolated nucleic acid encoding a
transport peptide comprising SEQ ID NO: 1.
16. The composition of claim 15, wherein the transport peptide
consists of SEQ ID NO: 1.
17. The composition of claim 15, wherein the nucleic acid comprises
5'-CGGCGCCCGCCTCGT-3' (SEQ ID NO: 7).
18. The composition of claim 15, further comprising a nucleic acid
encoding at least one cargo moiety selected from the group
consisting of a peptide; a protein; a biologically active compound;
a label; an imaging agent; a diagnostic agent; a therapeutic agent;
and a prophylactic agent.
19. A composition comprising an isolated nucleic acid encoding a
transport peptide comprising SEQ ID NO: 1, further comprising an
additional nucleic acid encoding at least one cargo moiety selected
from the group consisting of SEQ ID NOs: 3-6.
20. The composition of claim 19, which comprises a nucleic acid
encoding a transport construct selected from the group consisting
of SEQ ID NO: 1-SEQ ID NO: 3; SEQ ID NO: 1-SEQ ID NO: 4; SEQ ID NO:
1-SEQ ID NO: 5; SEQ ID NO: 1-SEQ ID NO: 6; SEQ ID NO: 3-SEQ ID NO:
1; SEQ ID NO: 4-SEQ ID NO: 1; SEQ ID NO: 5-SEQ ID NO: 1; and SEQ ID
NO: 6-SEQ ID NO: 1.
21. A vector comprising a nucleic acid encoding a transport peptide
comprising SEQ ID NO: 1.
22. The vector of claim 21, wherein the transport peptide consists
of SEQ ID NO: 1.
23. The vector of claim 21, further comprising transcriptional
activation elements that allow for the expression of the nucleic
acid encoding the transport peptide.
24. The vector of claim 21, further comprising a nucleic acid
encoding a cargo moiety in-frame with the nucleic acid encoding the
transport peptide.
25. An isolated host cell comprising exogenous nucleic acid
encoding a transport peptide comprising SEQ ID NO: 1.
26. The host cell of claim 25, wherein the nucleic acid is a vector
comprising (a) a nucleic acid encoding the transport peptide, and
(b) a nucleic acid encoding a cargo moiety in-frame with the
nucleic acid encoding the transport peptide.
27. The host cell of claim 26, further comprising transcriptional
activation elements that allow for the expression of the nucleic
acid of (a) and the nucleic acid of (b) in the host cell.
28. A method of delivering a cargo moiety to or into a target cell,
the method comprising contacting the target cell with a transport
construct, wherein the transport construct comprises a cargo moiety
linked to a transport peptide comprising SEQ ID NO: 1, whereby the
cargo moiety is delivered to or into the target cell.
29. The method of claim 28, wherein the cargo moiety is covalently
linked to the transport peptide through a linker or a chemical
bond.
30. The method of claim 29, wherein the linker comprises a
disulfide bond or wherein the chemical bond between the cargo
moiety and the transport peptide comprises a disulfide bond.
31. The method of claim 29, wherein the cargo moiety comprises a
peptide moiety, and wherein the transport peptide is covalently
linked through an amide bond to the N-terminus or C-terminus of the
peptide moiety of the cargo moiety.
32. The method of claim 28, wherein the transport peptide consists
of SEQ ID NO: 1.
33. The method of claim 28, wherein the cargo moiety is at least
one selected from the group consisting of a nucleic acid; peptide;
protein; oligosaccharide; lipid; glycolipid; lipoprotein; small
molecule compound; therapeutic drug; UV-vis, fluorescent or
radioactive label; imaging agent; diagnostic agent; prophylactic
agent; liposome and virus.
34. The method of claim 28, wherein the target cell comprises at
least one selected from the group consisting of an endothelial
cell, cardiac cell, immune cell, skeletal muscle cell and brain
cell.
35. The method of claim 28, wherein the cell is mammalian.
36. The method of claim 35, wherein the mammal is human.
37. A method of delivering a cargo moiety to or into a target cell
of a subject in need thereof, the method comprising administering
to the subject a therapeutically effective amount of a transport
construct, wherein the transport construct comprises the cargo
moiety linked to a transport peptide comprising SEQ ID NO: 1,
whereby the cargo moiety is delivered to or into the target cell of
the subject.
38. The method of claim 37, wherein the cargo moiety is covalently
linked to the transport peptide through a linker or a chemical
bond.
39. The method of claim 38, wherein the linker comprises a
disulfide bond or wherein the chemical bond between the cargo
moiety and the transport peptide comprises a disulfide bond.
40. The method of claim 38, wherein the cargo moiety comprises a
peptide moiety, and wherein the transport peptide is covalently
linked through an amide bond to the N-terminus or C-terminus of the
peptide moiety of the cargo moiety.
41. The method of claim 37, wherein the transport peptide consists
of SEQ ID NO: 1.
42. The method of claim 37, wherein the cargo moiety is at least
one selected from the group consisting of a nucleic acid; peptide;
protein; oligosaccharide; lipid; glycolipid; lipoprotein; small
molecule compound; therapeutic drug; UV-vis, fluorescent or
radioactive label; imaging agent; diagnostic agent; prophylactic
agent; liposome and virus.
43. The method of claim 37, wherein the target cell comprises at
least one selected from the group consisting of an endothelial
cell, cardiac cell, immune cell, skeletal muscle cell and brain
cell.
44. The method of claim 37, wherein the transport construct is
administered to the subject by at least one route selected from the
group consisting of oral, transmucosal, topical, transdermal,
intradermal, subcutaneous, ophthalmic, intravitreal,
subconjunctival, suprachoroidal, intracameral, inhalational,
intrabronchial, pulmonary, intravenous, intra-arterial,
intraduodenal, intravesical, parenteral, intrathecal, intramuscular
and intragastrical.
45. The method of claim 37, wherein the subject is a mammal.
46. The method of claim 45, wherein the mammal is human.
47. A method of delivering a cargo moiety to or into a target cell,
the method comprising contacting the target cell with a transport
construct, wherein the transport construct comprises a transport
peptide comprising SEQ ID NO:1 that is linked to a cargo moiety
comprising a sequence selected from the group consisting of SEQ ID
NOs: 3-6, whereby the cargo moiety is delivered to or into the
target cell.
48. The method of claim 47, wherein the transport peptide is
covalently linked to the cargo moiety through a linker or a
chemical bond.
49. The method of claim 48, wherein the linker comprises a
disulfide bond or wherein the chemical bond between the cargo
moiety and the transport peptide comprises a disulfide bond.
50. The method of claim 48, wherein the cargo moiety comprises a
peptide moiety, and wherein the transport peptide is covalently
linked through an amide bond to the N-terminus or C-terminus of the
peptide moiety of the cargo moiety.
51. The method of claim 47, wherein the transport construct
comprises at least one sequence selected from the group consisting
of SEQ ID NO: 1-SEQ ID NO: 3; SEQ ID NO: 1-SEQ ID NO: 4; SEQ ID NO:
1-SEQ ID NO: 5; SEQ ID NO: 1-SEQ ID NO: 6; SEQ ID NO: 3-SEQ ID NO:
1; SEQ ID NO: 4-SEQ ID NO: 1; SEQ ID NO: 5-SEQ ID NO: 1; and SEQ ID
NO: 6-SEQ ID NO: 1.
52. The method of claim 47, wherein the target cell comprises at
least one selected from the group consisting of an endothelial
cell, cardiac cell, immune cell, skeletal muscle cell and brain
cell.
53. The method of claim 47, wherein the cell is mammalian.
54. The method of claim 53, wherein the mammal is human.
55. A method of delivering a cargo moiety to or into a target cell
of a subject in need thereof, the method comprising administering
to the subject a therapeutically effective amount of a transport
construct, wherein the transport construct comprises a transport
peptide comprising SEQ ID NO:1 that is linked to a cargo moiety
comprising a sequence selected from the group consisting of SEQ ID
NOs: 3-6, whereby the cargo moiety is delivered to or into the
target cell of the subject.
56. The method of claim 55, wherein the transport peptide is
covalently linked to the cargo moiety through a linker or a
chemical bond.
57. The method of claim 56, wherein the linker comprises a
disulfide bond or wherein the chemical bond between the cargo
moiety and the transport peptide comprises a disulfide bond.
58. The method of claim 56, wherein the cargo moiety comprises a
peptide moiety, and wherein the transport peptide is covalently
linked through an amide bond to the N-terminus or C-terminus of the
peptide moiety of the cargo moiety.
59. The method of claim 55, wherein the transport construct
comprises at least one sequence selected from the group consisting
of SEQ ID NO: 1-SEQ ID NO: 3; SEQ ID NO: 1-SEQ ID NO: 4; SEQ ID NO:
1-SEQ ID NO: 5; SEQ ID NO: 1-SEQ ID NO: 6; SEQ ID NO: 3-SEQ ID NO:
1; SEQ ID NO: 4-SEQ ID NO: 1; SEQ ID NO: 5-SEQ ID NO: 1; and SEQ ID
NO: 6-SEQ ID NO: 1.
60. The method of claim 55, wherein the target cell comprises at
least one selected from the group consisting of an endothelial
cell, cardiac cell, immune cell, skeletal muscle cell and brain
cell.
61. The method of claim 55, wherein the transport construct is
administered to the subject by at least one route selected from the
group consisting of oral, transmucosal, topical, transdermal,
intradermal, subcutaneous, ophthalmic, intravitreal,
subconjunctival, suprachoroidal, intracameral, inhalational,
intrabronchial, pulmonary, intravenous, intra-arterial,
intraduodenal, intravesical, parenteral, intrathecal, intramuscular
and intragastrical.
62. The method of claim 55, wherein the subject is a mammal.
63. The method of claim 62, wherein the mammal is human.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation of, and claims
priority to, U.S. patent application Ser. No. 15/036,849, filed May
16, 2016, now allowed, which is a 35 U.S.C. .sctn. 371 national
phase application from, and claims priority to, International
Application No. PCT/US2014/066619, filed Nov. 20, 2014, and
published under PCT Article 21(2) in English, which claims priority
under 35 U.S.C. .sctn. 119(e) to U.S. Provisional Patent
Application No. 61/908,963, filed Nov. 26, 2013, all of which
applications are incorporated herein by reference in their
entireties.
BACKGROUND OF THE INVENTION
[0003] The cell membrane (also known as the plasma membrane or
cytoplasmic membrane) is a biological membrane that separates the
interior of the cell from the outside environment, protecting the
cell from its surroundings. The membrane comprises a phospholipid
bilayer with embedded proteins, and is involved in cellular
processes such as cell adhesion, ion conductivity and cell
signaling.
[0004] The cell membrane controls the movement of substances in and
out of cells and is selectively permeable to ions and organic
molecules. The movement of substances across the membrane may be
passive (i.e., occurring without the input of cellular energy) or
active (i.e., requiring the cell to expend energy in transporting
it). The cell membrane thus works as a selective filter, employing
transport mechanisms such as passive osmosis and diffusion,
transmembrane protein channels transportation, endocytosis and
exocytosis.
[0005] Intracellular delivery of biologically active compounds is
challenging because the cell membrane is remarkably impermeable to
extracellular polar compounds. There is thus much interest in
identifying novel cell-permeable peptides ("CPPs") that can act as
"Trojan horses" for carrying cargo molecules inside living cells.
CPPs have been employed in intracellular delivery of
oligonucleotides (Astriab-Fisher et al., 2000, Biochem. Pharmacol.
60:83-90; Eguchi et al., 2001, J. Biol. Chem. 276:26204-26210),
plasmids (Morris et al., 1999, Nucleic Acids Res. 27:3510-3517),
viruses (Grafton et al., 2003, Nat. Med. 9:357-362), peptides
(Gratton et al., 2003, Nat. Med. 9:357-362; Soomets et al., 2000,
Biochim. Biophys. Acta 1467:165-176) and fluorophores (Bucci et
al., 2000, Nat. Med. 6:1362-1367). The Antennapedia homeodomain
("AP"; a 16-amino acid peptide, which is a Drosophila transcription
factor), as well as the HIV transactivator of transcription ("TAT";
15 amino acids) are amongst the first CPPs described, along with
more recently described CPP sequences, such as poly-Arginine
(Arg.sub.7 or Arg.sub.9) and C105Y (a 17-amino acid peptide).
[0006] The capacity of CPPs to translocate cargo into cells could
make them attractive delivery agents for cell-impermeable
therapeutic compounds. However, the therapeutic effect, kinetics,
safety profile and specificity of CPPs in humans are still unknown.
Novel target-engineered CPPs with enhanced internalization
capabilities, enhanced overall therapeutic efficacy and safety,
minimal peptide elimination/degradation and high therapeutic
activity/cost ratio are required.
[0007] Caveolins are cholesterol binding proteins that may regulate
signal transduction pathways (Smart et al., 1999, Mol. Cell. Biol.
19:7289-7304; Kurzchalia & Parton, 1999, Curr. Opin. Cell.
Biol. 11:424-431). Recent studies have focused on their subcellular
trafficking and regulation of endothelial nitric oxide synthase
(eNOS). eNOS-derived NO is necessary for the maintenance of
systemic blood pressure, vascular remodeling, angiogenesis and
wound healing (Huang et al., 1995, Nature 377:239-242; Murohara et
al., 1998, J. Clin. Invest. 101:2567-2578; Rudic et al., 1998, J.
Clin. Invest. 101:731-736; Lee et al., 1999, Am. J. Physiol.
277:HI600-1608). eNOS can physically interact with caveolin-1 and
caveolin-3 by binding to their putative scaffolding domain located
between residues 82-101 (Li et al., 1996, J. Biol. Chem.
271:29182-29190), and this interaction renders eNOS in its "less
active" state (Garcia-Cardena et al., 1997, J. Biol: Chem.
272:25437-25440; Ju et al., 1997, J. Biol. Chem. 272:18522-18525;
Michel et al., 1997, J. Biol. Chem. 272:25907-25912). Consistent
with the model of caveolin as a negative regulator of eNOS,
peptides derived from the scaffolding domain of caveolin-1 disrupt
the binding of eNOS to caveolin and inhibit NOS activity in a dose
dependent manner in vitro (IC.sub.50=1-3 .mu.M) by slowing electron
flux from the reductase to the oxygenase domain of NOS
(Garcia-Cardena et al., 1997, J. Biol. Chem. 272:25437-25440; Ju et
al., 1997, J. Biol. Chem. 272:18522-18525; Ghosh et al., 1998, J.
Biol. Chem. 273:22267-22271).
[0008] There is a need in the art to identify novel molecules that
efficiently penetrate cell membranes. Such molecules would be
useful in promoting the delivery of cargo moieties, such as
therapeutic agents, nucleic acids, peptides, saccharides, lipids,
liposomes and such, across the cell membrane. The present invention
satisfies this unmet need.
BRIEF SUMMARY OF THE INVENTION
[0009] The invention provides an isolated transport peptide, or a
salt or solvate thereof, comprising the amino acid sequence RRPPR
(SEQ ID NO: 1).
[0010] The invention further provides an isolated transport
construct, or a salt or solvate thereof, comprising a transport
peptide comprising SEQ ID NO: 1 linked to a cargo moiety.
[0011] The invention further provides an isolated transport
construct, or a salt or solvate thereof, comprising a transport
peptide comprising SEQ ID NO:1 that is linked to a cargo moiety
comprising the sequence selected from the group consisting of SEQ
ID NOs: 3-6.
[0012] The invention further provides a composition comprising an
isolated nucleic acid encoding a transport peptide comprising SEQ
ID NO: 1.
[0013] The invention further provides a composition comprising an
isolated nucleic acid encoding a transport peptide comprising SEQ
ID NO: 1, further comprising an additional nucleic acid encoding at
least one cargo moiety selected from the group consisting of SEQ ID
NOs: 3-6.
[0014] The invention further provides a vector comprising a nucleic
acid encoding a transport peptide comprising SEQ ID NO: 1.
[0015] The invention further provides an isolated host cell
comprising exogenous nucleic acid encoding a transport peptide
comprising SEQ ID NO: 1.
[0016] The invention further provides a method of delivering a
cargo moiety (in)to a target cell.
[0017] The invention further provides a method of delivering a
cargo moiety (in)to a target cell of a subject in need thereof.
[0018] In certain embodiments, the transport peptide consists of
SEQ ID NO: 1. In other embodiments, the transport peptide and/or
construct is/are part of a pharmaceutical composition further
comprising a pharmaceutically acceptable carrier. In yet other
embodiments, the cargo moiety is at least one selected from the
group consisting of a nucleic acid; peptide; protein;
oligosaccharide; lipid; glycolipid; lipoprotein; small molecule
compound; therapeutic drug; UV-vis, fluorescent or radioactive
label; imaging agent; diagnostic agent; prophylactic agent;
liposome and virus. In yet other embodiments, the nucleic acid
comprises 5'-CGGCGCCCGCCTCGT-3' (SEQ ID NO: 7). In yet other
embodiments, the composition comprises a nucleic acid encoding a
transport construct selected from the group consisting of SEQ ID
NO: 1-SEQ ID NO: 3; SEQ ID NO: 1-SEQ ID NO: 4; SEQ ID NO: 1-SEQ ID
NO: 5; SEQ ID NO: 1-SEQ ID NO: 6; SEQ ID NO: 3-SEQ ID NO: 1; SEQ ID
NO: 4-SEQ ID NO: 1; SEQ ID NO: 5-SEQ ID NO: 1; and SEQ ID NO: 6-SEQ
ID NO: 1. In yet other embodiments, the composition further
comprises a nucleic acid encoding at least one cargo moiety
selected from the group consisting of a peptide; a protein; a
biologically active compound; a label; an imaging agent; a
diagnostic agent; a therapeutic agent; and a prophylactic
agent.
[0019] In certain embodiments, the cargo moiety is covalently
linked to the transport peptide through a linker or a chemical
bond. In other embodiments, the linker comprises a disulfide bond,
or the chemical bond between the cargo moiety and the transport
peptide comprises a disulfide bond. In yet other embodiments, the
cargo moiety comprises a peptide moiety. In yet other embodiments,
the cargo moiety comprises a peptide or protein. In yet other
embodiments, the transport peptide is covalently linked through an
amide bond to the N-terminus of the peptide moiety of the cargo
moiety. In yet other embodiments, the transport peptide is
covalently linked through an amide bond to the C-terminus of the
peptide moiety of the cargo moiety.
[0020] In certain embodiments, the transport construct comprises at
least one sequence selected from the group consisting of SEQ ID NO:
1-SEQ ID NO: 3; SEQ ID NO: 1-SEQ ID NO: 4; SEQ ID NO: 1-SEQ ID NO:
5; SEQ ID NO: 1-SEQ ID NO: 6; SEQ ID NO: 3-SEQ ID NO: 1; SEQ ID NO:
4-SEQ ID NO: 1; SEQ ID NO: 5-SEQ ID NO: 1; and SEQ ID NO: 6-SEQ ID
NO: 1. In other embodiments, the transport construct is selected
from the group consisting of SEQ ID NO: 1-SEQ ID NO: 3; SEQ ID NO:
1-SEQ ID NO: 4; SEQ ID NO: 1-SEQ ID NO: 5; SEQ ID NO: 1-SEQ ID NO:
6; SEQ ID NO: 3-SEQ ID NO: 1; SEQ ID NO: 4-SEQ ID NO: 1; SEQ ID NO:
5-SEQ ID NO: 1; and SEQ ID NO: 6-SEQ ID NO: 1.
[0021] In certain embodiments, the vector further comprises
transcriptional activation elements that allow for the expression
of the nucleic acid encoding the transport peptide. In other
embodiments, the vector comprises a nucleic acid encoding a cargo
moiety in-frame with the nucleic acid encoding the transport
peptide. In yet other embodiments, the nucleic acid is a vector
comprising (a) a nucleic acid encoding the transport peptide, and
(b) a nucleic acid encoding a cargo moiety in-frame with the
nucleic acid encoding the transport peptide. In yet other
embodiments, the host cell further comprises transcriptional
activation elements that allow for the expression of the nucleic
acid of (a) and the nucleic acid of (b) in the host cell.
[0022] In certain embodiments, the transport peptide binds to a
target cell and/or crosses a cell membrane. In other embodiments,
the transport construct binds to a target cell and/or crosses a
cell membrane. In yet other embodiments, the target cell comprises
at least one selected from the group consisting of an endothelial
cell, cardiac cell, immune cell, skeletal muscle cell and brain
cell. In yet other embodiments, the cell is mammalian. In yet other
embodiments, the mammal is human.
[0023] In certain embodiments, the method comprises contacting the
target cell with a transport construct, wherein the transport
construct comprises a cargo moiety linked a transport peptide
comprising SEQ ID NO: 1, whereby the cargo moiety is delivered to
or into the target cell.
[0024] In certain embodiments, the method comprises contacting the
target cell with a transport construct, wherein the transport
construct comprises a transport peptide comprising SEQ ID NO:1
linked to a cargo moiety comprising a sequence selected from the
group consisting of SEQ ID NOs: 3-6, whereby the cargo moiety is
delivered to or into the target cell.
[0025] In certain embodiments, the method comprises administering
to the subject a therapeutically effective amount of a transport
construct, wherein the transport construct comprises the cargo
moiety linked to a transport peptide comprising SEQ ID NO: 1,
whereby the cargo moiety is delivered to or into the target cell of
the subject.
[0026] In certain embodiments, the method comprises administering
to the subject a therapeutically effective amount of a transport
construct, wherein the transport construct comprises a transport
peptide comprising SEQ ID NO:1 that is linked to a cargo moiety
comprising a sequence selected from the group consisting of SEQ ID
NOs: 3-6, whereby the cargo moiety is delivered to or into the
target cell of the subject.
[0027] In certain embodiments, a compound and/or composition of the
invention is/are administered to the subject by at least one route
selected from the group consisting of oral, transmucosal, topical,
transdermal, intradermal, subcutaneous, ophthalmic, intravitreal,
subconjunctival, suprachoroidal, intracameral, inhalational,
intrabronchial, pulmonary, intravenous, intra-arterial,
intraduodenal, intravesical, parenteral, intrathecal, intramuscular
and intragastrical.
BRIEF DESCRIPTION OF THE DRAWINGS
[0028] The following detailed description of specific embodiments
of the invention will be better understood when read in conjunction
with the appended drawings. For the purpose of illustrating the
invention, specific embodiments are shown in the drawings. It
should be understood, however, that the invention is not limited to
the precise arrangements and instrumentalities of the embodiments
shown in the drawings.
[0029] FIG. 1 is a graph illustrating the exponential enrichment of
cell-permeable phage during biopanning. The percentages of
recovered phage from Table 1 are plotted for the six rounds of
biopanning in endothelial cells ("EC"). Exponential correlation is
established with a R.sup.2 value of 0.975.
[0030] FIGS. 2A-2D are a series of bar graphs illustrating the
finding that Endo5-Cav is more potent than AP-Cav at blocking
VEGF-induced NO release. FIG. 2A: Endo5-Cav completely blocked
VEGF-induced NO release. Cultured BAEC were pretreated for 6 hours
with the indicated peptides (10.sup.-5 M) and stimulated with VEGF
(10.sup.-9 M) for 30 min as indicated. *P<0.05 compared with
vehicle, and .dagger.P<0.05 compared with AP-Cav+VEGF. n=4 in
triplicate. FIG. 2B: AP-Cav and Endo5-Cav showed a dose-dependent
effect. Cultured BAEC were pretreated with peptides
(1-50.times.10.sup.-6 M) for 6 hours and stimulated with VEGF as
described in FIG. 2A. *P<0.05 compared with vehicle, and
.dagger.P<0.05 compared with AP-Cav+VEGF. n=4 in duplicate. FIG.
2C: AP-Cav and Endo5-Cav showed time dependent effects. BAEC were
treated with peptides (10.sup.-5 M) for 1, 2, 4 or 6 hours, and
stimulated with VEGF as described in FIG. 2A. *P<0.05 compared
with vehicle, and .dagger.P<0.05 compared with AP-Cav+VEGF. n=4
in duplicate. FIG. 2D: Optimization of both the cell-penetrating
sequence and Cav domain of AP-Cav led to a shorter, more potent
eNOS inhibitor. Substitution of AP to Endo5 and shortening of
Cav(82-101) to CavAB(82-95) (EndoS-CavAB; 10.sup.-5 M) completely
blocked VEGF-induced NO release, whereas Endo5-CavAB
(2.times.10.sup.-6 M), a much shorter peptide at a lesser dose, had
a similar effect to AP-Cav (10.sup.-5 M).
[0031] FIGS. 3A-3B illustrate the finding that Endo5-Cav blocks
Evans blue extravasation in vivo. FIG. 3A: Pretreatment of mice
with AP-Cav (1 mg/kg) or Endo5-Cav (same dose on a molar basis) for
1 hour prevented mustard oil-induced increase in vascular
permeability (right ear; 30 min), whereas control peptides had no
significant effect. Left ears were painted with mineral oil alone
(vehicle) and considered as baseline control. Mice were
pre-injected with Evans blue. *P<0.05 compared with control
peptide, and .dagger.P<0.05 compared with AP-Cav+mustard oil.
n=6 or 8 per group in duplicate. FIG. 3B: Representative values for
the data presented in FIG. 3A are illustrated.
[0032] FIGS. 4A-4B are a series of graphs illustrating the finding
that Endo5 is internalized faster than AP in cultured endothelial
cells. FIG. 4A: Fluorescence readouts for similar concentration of
rhodamine-AP (rhod-AP) and carboxyfluorescein-Endo5 (cFluo-Endo5)
dissolved in the same cell lysis solution used in FIG. 4B were
performed to confirm the linearity between peptide concentration in
solution and fluorescence values. Peptides were used separately to
prevent interference. FIG. 4B: Carboxyfluorescein-Endo5 rate of
internalization is greater than that of rhodamine-AP. Cultured BAEC
were incubated for 1, 2, 4 or 6 hours with individual peptides,
acid washed, rinsed, trypsinized, lysed and total internal
fluorescence was determined and converted to moles of peptides per
10.sup.6 by using a standard curve. Cells incubated with peptides
for 5 min and treated as described were used as background for
non-internalized staining.
[0033] FIGS. 5A-5C illustrate the finding that internalization of
Endo5 and AP uses overlapping cellular pathways in endothelial
cells. FIG. 5A: Cultured HUVEC were treated with
carboxyfluorescein-Endo5 (green) or rhodamine-AP (red; 10.sup.-5 M)
for 1 hour (pulse), rinsed and live imaging in unfixed cells was
performed using an epifluorescence microscope. Note the punctate
staining with both peptides and the absence of nuclear staining
(oval-shaped dark zone). Merged images showed localization (yellow)
between both peptides. Representative cells shown. FIG. 5B: After
treatment described in FIG. 5A, peptide localization was chased for
two hours in live HUVEC and visualized. Colocalization (yellow)
between both peptides was still observable. FIG. 5C: Endo5 and AP
prevented AP-Cav and Endo5-Cav inhibition of VEGF-induced NO
release. Cultured BAEC were pretreated with either AP or Endo5
(5.times.10.sup.-5 M) and incubated for 6 h with either AP-Cav or
Endo5-Cav (10.sup.-5 M) and stimulated with VEGF as described in
FIG. 2. *P<0.05 compared with vehicle n=6 per group in
triplicate.
DETAILED DESCRIPTION OF THE INVENTION
[0034] The invention relates in part to the unexpected
identification of Endo5 (RRPPR; SEQ ID NO: 1), a short five-amino
acid peptide, as a cell-permeable peptide (CPP).
[0035] In certain embodiments, Endo5 is a transport peptide that
crosses the cell membrane. In other embodiments, once a cargo
moiety is linked to Endo5, the resulting construct crosses the cell
membrane more efficiently than the cargo moiety itself. In certain
embodiments, the cargo moiety is selected from the group consisting
of a nucleic acid; peptide; protein; oligosaccharide; lipid;
glycolipid; lipoprotein; small molecule compound; therapeutic drug;
UV-vis, fluorescent or radioactive label; imaging agent; diagnostic
agent; prophylactic agent; liposome and virus. In other
embodiments, the cargo moiety is linked to the transport peptide
through a covalent or non-covalent linkage.
[0036] As demonstrated herein, Endo5, a short pentapeptide, was
unexpectedly isolated using a phage display library-based approach.
Endo5 was selected for its capacity to increase phage
internalization in human endothelial cells. Functional analyses
revealed that Endo5-Cav was more potent than AP-Cav at inhibiting
vascular endothelial growth factor (VEGF)-induced nitric oxide
release in endothelial cells in vitro and permeability in vivo.
Pharmacokinetic and competition studies showed that Endo5 was
internalized by endothelial cells at a greater rate than AP, and
that Endo5-Cav activities were competitively inhibited by AP,
providing evidence for the similarity of Endo5 and AP uptake
pathways. As supported by the data reported herein, Endo5 is not
only the shortest CPP sequence known at the time of the invention,
but also as the first CPP engineered specifically for high
internalization rates in endothelial cells.
Definitions
[0037] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, non-limiting methods and materials are described.
[0038] The articles "a" and "an" are used herein to refer to one or
to more than one (i.e., to at least one) of the grammatical object
of the article. By way of example, "an element" means one element
or more than one element.
[0039] As used herein, the term "about" when referring to a
measurable value such as an amount, a temporal duration, and the
like, is meant to encompass variations of .+-.20% or .+-.10%, more
specifically .+-.5%, even more specifically .+-.1%, and still more
specifically .+-.0.1% from the specified value, as such variations
are appropriate to perform the disclosed methods.
[0040] A disease or disorder is "alleviated" if the severity of a
symptom of the disease or disorder, the frequency with which such a
symptom is experienced by a patient, or both, is reduced.
[0041] The term "amino acid sequence variant" refers to
polypeptides having amino acid sequences that differ to some extent
from a native sequence polypeptide. Ordinarily, amino acid sequence
variants will possess at least about 70% homology, at least about
80% homology, at least about 90% homology, at least about 95%
homology, at least about 96% homology, at least about 97% homology,
at least about 98% homology, or at least about 99% homology to the
native polypeptide. The amino acid sequence variants possess
substitutions, deletions, and/or insertions at certain positions
within the amino acid sequence of the native amino acid
sequence.
[0042] As used herein, the term "AP" refers to the Antennapedia
homeodomain (a 16-amino acid peptide that is a Drosophila
transcription factor), which is the peptide of SEQ ID NO: 2 or a
salt or solvate thereof.
[0043] As used herein, the term "BAEC" refers to bovine aorta
endothelial cell(s).
[0044] As used herein, the term "binding" refers to the adherence
of molecules to one another, such as, but not limited to, enzymes
to substrates, antibodies to antigens, DNA strands to their
complementary strands. Binding occurs because the shape and
chemical nature of parts of the molecule surfaces are
complementary. A common model is the "lock-and-key" used to
describe how enzymes fit around their substrate. In a non-limiting
example, the binding of the caveolin protein may occur at one or
more domains of eNOS, such as, but not limited to, the oxygenase
domain of eNOS and/or the reductase domain of eNOS.
[0045] As used herein, the term "caveolin scaffolding domain"
refers to domains inclusive of putative scaffolding domains of any
caveolin protein. Thus, the term as used herein is not limited to
putative scaffolding domains. The complete mRNA sequence of human
Cav-1 may be found at GenBank Accession No. BAG70230.1 (SEQ ID NO:
3). The complete protein code for human Cav-3 may be found at
GenBank Accession No. AAC39758.1 (SEQ ID NO: 4).
[0046] Examples of caveolin scaffolding domains include, but are
not limited to, the following: amino acids 82-101 of human
caveolin-1 (.sup.82DGIWKASFTTFTVTKYWFYR.sup.101) (SEQ ID NO: 5) or
equivalents thereof amino acids 82-95 of human caveolin-1
(.sup.82DGIWKASFTTFTVT.sup.95) (SEQ ID NO: 6) or equivalents
thereof.
[0047] As used herein, the terms "conservative variation" or
"conservative substitution" as used herein refers to the
replacement of an amino acid residue by another, biologically
similar residue. Conservative variations or substitutions are not
likely to change the shape of the peptide chain. Examples of
conservative variations, or substitutions, include the replacement
of one hydrophobic residue such as isoleucine, valine, leucine or
methionine for another, or the substitution of one polar residue
for another, such as the substitution of arginine for lysine,
glutamic for aspartic acid, or glutamine for asparagine.
[0048] A "disease" is a state of health of an animal wherein the
animal cannot maintain homeostasis, and wherein if the disease is
not ameliorated then the animal's health continues to
deteriorate.
[0049] A "disorder" in an animal is a state of health in which the
animal is able to maintain homeostasis, but in which the animal's
state of health is less favorable than it would be in the absence
of the disorder. Left untreated, a disorder does not necessarily
cause a further decrease in the animal's state of health.
[0050] As used herein, the term "domain" refers to a part of a
molecule or structure that shares common physicochemical features,
such as, but not limited to, hydrophobic, polar, globular and
helical domains or properties. Specific examples of binding domains
include, but are not limited to, DNA binding domains and ATP
binding domains.
[0051] As used herein, the term "EC" refers to endothelial
cell(s).
[0052] As used herein, the term "Endo5" refers to the peptide of
SEQ ID NO: 1 or a salt or solvate thereof.
[0053] As used herein, the term "Evans blue" refers to any salt or
solvate of
(6E,6'E)-6,6-[(3,3'-dimethylbiphenyl-4,4'-diyl)di(1E)hydrazin-2-yl-1-y-
lidene]bis(4-amino-5-oxo-5,6-dihydronaphthalene-1,3-disulfonate).
[0054] As used herein, the term "heterologous peptide" refers to
any peptide, polypeptide or protein whose sequence is selected in
such a way that the product of the fusion of this sequence with the
membrane translocation domain has a sequence different from the
wild-type sequence flanking any membrane translocation domain.
[0055] As used herein, the term "membrane translocation domain"
refers to a peptide capable of permeating the membrane of a cell
and which is used to transport attached peptides into a cell in
vivo.
[0056] As used herein, the term "patient," "individual" or
"subject" refers to a human or a non-human mammal. Non-human
mammals include, for example, livestock and pets, such as ovine,
bovine, porcine, canine, feline and murine mammals. In certain
embodiments, the patient, individual or subject is human.
[0057] As used herein, the term "peptide" typically refers to short
polypeptides. Conventional notation is used herein to represent
polypeptide sequences: the left-hand end of a polypeptide sequence
is the amino-terminus, and the right-hand end of a polypeptide
sequence is the carboxyl-terminus.
[0058] As used herein, the term "pharmaceutical composition" or
"composition" refers to a mixture of at least one compound useful
within the invention with a pharmaceutically acceptable carrier.
The pharmaceutical composition facilitates administration of the
compound to a patient. Multiple techniques of administering a
compound exist in the art including, but not limited to,
intravenous, oral, aerosol, inhalational, rectal, vaginal,
transdermal, intranasal, buccal, sublingual, parenteral,
intrathecal, intragastrical, ophthalmic, pulmonary and topical
administration. In certain embodiments, routes of administration
include transdermal, transmucosal (e.g., sublingual, lingual,
(trans)buccal, (trans)urethral, vaginal (e.g., trans- and
perivaginally), (intra)nasal and (trans)rectal), intravesical,
intrapulmonary, intraduodenal, intragastrical, intrathecal,
subcutaneous, intramuscular, intradermal, intra-arterial,
intravenous, intrabronchial, inhalation, pleural, peritoneal,
subcutaneous, epidural, otic, intraocular, and/or topical
administration
[0059] As used herein, the term "pharmaceutically acceptable"
refers to a material, such as a carrier or diluent, which does not
abrogate the biological activity or properties of the compound, and
is relatively non-toxic, i.e., the material may be administered to
an individual without causing undesirable biological effects or
interacting in a deleterious manner with any of the components of
the composition in which it is contained.
[0060] As used herein, the term "pharmaceutically acceptable
carrier" means a pharmaceutically acceptable material, composition
or carrier, such as a liquid or solid filler, stabilizer,
dispersing agent, suspending agent, diluent, excipient, thickening
agent, solvent or encapsulating material, involved in carrying or
transporting a compound useful within the invention within or to
the patient such that it may perform its intended function.
Typically, such constructs are carried or transported from one
organ, or portion of the body, to another organ, or portion of the
body. Each carrier must be "acceptable" in the sense of being
compatible with the other ingredients of the formulation, including
the compound useful within the invention, and not injurious to the
patient. Some examples of materials that may serve as
pharmaceutically acceptable carriers include: sugars, such as
lactose, glucose and sucrose; starches, such as corn starch and
potato starch; cellulose, and its derivatives, such as sodium
carboxymethyl cellulose, ethyl cellulose and cellulose acetate;
powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa
butter and suppository waxes; oils, such as peanut oil, cottonseed
oil, safflower oil, sesame oil, olive oil, corn oil and soybean
oil; glycols, such as propylene glycol; polyols, such as glycerin,
sorbitol, mannitol and polyethylene glycol; esters, such as ethyl
oleate and ethyl laurate; agar; buffering agents, such as magnesium
hydroxide and aluminum hydroxide; surface active agents; alginic
acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl
alcohol; phosphate buffer solutions; and other non-toxic compatible
substances employed in pharmaceutical formulations. As used herein,
"pharmaceutically acceptable carrier" also includes any and all
coatings, antibacterial and antifungal agents, and absorption
delaying agents, and the like that are compatible with the activity
of the compound useful within the invention, and are
physiologically acceptable to the patient. Supplementary active
compounds may also be incorporated into the compositions. The
"pharmaceutically acceptable carrier" may further include a
pharmaceutically acceptable salt of the compound useful within the
invention. Other additional ingredients that may be included in the
pharmaceutical compositions used in the practice of the invention
are known in the art and described, for example in Remington's
Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985,
Easton, Pa.), which is incorporated herein by reference.
[0061] As used herein, the language "pharmaceutically acceptable
salt" refers to a salt of the administered compound prepared from
pharmaceutically acceptable non-toxic acids and bases, including
inorganic acids, inorganic bases, organic acids, inorganic bases,
solvates, hydrates, and clathrates thereof. Suitable
pharmaceutically acceptable acid addition salts may be prepared
from an inorganic acid or from an organic acid. Examples of
inorganic acids include sulfate, hydrogen sulfate, hydrochloric,
hydrobromic, hydriodic, nitric, carbonic, sulfuric, and phosphoric
acids (including hydrogen phosphate and dihydrogen phosphate).
Appropriate organic acids may be selected from aliphatic,
cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic and
sulfonic classes of organic acids, examples of which include
formic, acetic, propionic, succinic, glycolic, gluconic, lactic,
malic, tartaric, citric, ascorbic, glucuronic, maleic, fumaric,
pyruvic, aspartic, glutamic, benzoic, anthranilic,
4-hydroxybenzoic, phenylacetic, mandelic, embonic (pamoic),
methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic,
trifluoromethanesulfonic, 2-hydroxyethanesulfonic,
p-toluenesulfonic, sulfanilic, cyclohexylaminosulfonic, stearic,
alginic, O-hydroxybutyric, salicylic, galactaric and galacturonic
acid. Suitable pharmaceutically acceptable base addition salts of
compounds of the invention include, for example, metallic salts
including ammonium salts and alkali metal, alkaline earth metal and
transition metal salts such as, for example, calcium, magnesium,
potassium, sodium and zinc salts. Pharmaceutically acceptable base
addition salts also include organic salts made from basic amines
such as, for example, N,N'-dibenzylethylene-diamine,
chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine
(N-methylglucamine) and procaine. All of these salts may be
prepared from the corresponding compound by reacting, for example,
the appropriate acid or base with the compound.
[0062] As used herein, the terms "pharmaceutically effective
amount" and "effective amount" and "therapeutically effective
amount" refer to a nontoxic but sufficient amount of an agent to
provide the desired biological result. That result may be reduction
and/or alleviation of the signs, symptoms, or causes of a disease,
or any other desired alteration of a biological system. An
appropriate therapeutic amount in any individual case may be
determined by one of ordinary skill in the art using routine
experimentation.
[0063] As used herein, the term "PNA" refers to a peptide nucleic
acid.
[0064] As used herein, the term "polypeptide" refers to a polymer
composed of amino acid residues, related naturally occurring
structural variants, and synthetic non-naturally occurring analogs
thereof linked via peptide (or amide) bonds. Synthetic polypeptides
may be synthesized, for example, using an automated polypeptide
synthesizer.
[0065] As used herein, the term "prevent" or "prevention" means no
disorder or disease development if none had occurred, or no further
disorder or disease development if there had already been
development of the disorder or disease. Also considered is the
ability of one to prevent some or all of the symptoms associated
with the disorder or disease.
[0066] As used herein, the term "protein" typically refers to large
polypeptides.
[0067] As used herein, the term "RHMVEC" refers to rat heart
microvascular endothelial cell(s).
[0068] As used herein, the term `solvate` refers to a complex
between a molecule and a solvent molecule, which may exist in
solution or in solid phase. In certain embodiments, the solvent
comprises at least one selected from the group consisting of water,
methanol, ethanol, n-propanol, 2-propanol, DMSO, DMF, ethyl ether,
acetone and pyridine.
[0069] As used herein, the term "transport construct" refers to a
construct that crosses the cell membrane, wherein the construct
comprises the transport peptide and at least one cargo moiety,
wherein the cargo moiety crosses the cell membrane at a lower rate
or to a lower degree than the transport construct. In certain
embodiments, the cargo moiety is selected from the group consisting
of a nucleic acid; peptide; protein; oligosaccharide; lipid;
glycolipid; lipoprotein; small molecule compound; therapeutic drug;
UV-vis, fluorescent or radioactive label; imaging agent; diagnostic
agent; prophylactic agent; liposome and virus. In other
embodiments, the cargo moiety is linked to the transport peptide
through a covalent or non-covalent linkage.
[0070] As used herein, the term "transport peptide" or "CPP" refers
to a cell-permeable peptide, which is defined as a peptide capable
of permeating and/or crossing a cell membrane.
[0071] As used herein, the term "treatment" or "treating" is
defined as the application or administration of a therapeutic
agent, i.e., a compound useful within the invention (alone or in
combination with another pharmaceutical agent), to a patient, or
application or administration of a therapeutic agent to an isolated
tissue or cell line from a patient (e.g., for diagnosis or ex vivo
applications), who has a disease or disorder, a symptom of a
disease or disorder or the potential to develop a disease or
disorder, with the purpose to cure, heal, alleviate, relieve,
alter, remedy, ameliorate, improve or affect the disease or
disorder, the symptoms of the disease or disorder, or the potential
to develop the disease or disorder. Such treatments may be
specifically tailored or modified, based on knowledge obtained from
the field of pharmacogenomics.
[0072] Ranges: throughout this disclosure, various aspects of the
invention can be presented in a range format. It should be
understood that the description in range format is merely for
convenience and brevity and should not be construed as an
inflexible limitation on the scope of the invention. Accordingly,
the description of a range should be considered to have
specifically disclosed all the possible subranges as well as
individual numerical values within that range. For example,
description of a range such as from 1 to 6 should be considered to
have specifically disclosed subranges such as from 1 to 3, from 1
to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as
well as individual numbers within that range, for example, 1, 2,
2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of
the range.
DESCRIPTION
[0073] The invention relates to the unexpected finding that Endo5,
a short pentapeptide (RRPPR), is a very potent CPP. As described
herein, Endo5 was isolated though a competitive selection process
for its capacity to be quickly internalized by vascular endothelial
cells. Endo5 was further shown to increase cargo uptake by cells
and increase therapeutic activity of a cargo to which it is
conjugated.
[0074] As described herein, Endo5 was the most highly enriched
randomly generated peptide when expressed at the surface of phages
selected for the ability to be quickly internalized by endothelial
cells. The maximum potency of Endo5-Cav at inhibiting VEGF-induced
NO release was greater than that of AP-Cav on a similar molar
basis. Further, despite its smaller size (Endo5 is a 5-mer peptide
vs AP is a 16-mer acid peptide), the rate of uptake of Endo5 was
three times greater than that of AP. Endo5-Cav was found to be more
potent than AP-Cav at inhibiting vascular permeability in vivo.
[0075] Mechanistically, the cellular pathways involved in Endo5
uptake appear similar to that of AP. This is supported by the
following findings: significant co-localization between Endo5 and
AP during initial endocytosis and intracellular distribution; the
competitive inhibition of Endo5-Cav effect by AP, and the
competitive inhibition of AP-Cav activity by Endo5. The findings
reported herein provide evidence for Endo5 higher "internalization
efficiency per amino acid" abilities compared to the
well-established AP.
[0076] The rationale for designing a potent CPP for an endothelial
cell resides not only in the various diseases characterized by
aberrant endothelial cell activity, but also from the strategic
localization of endothelial cells between the blood and underlying
tissues. Following absorption, drug distribution through the
vascular compartment may be quickly impaired though elimination and
degradation. These normal drug inactivation mechanisms may be
offset by rapid internalization at the site of action. Evidence of
the magnitude of Endo5 internalization in endothelial cells,
compared to the pool of randomly generated CPP the present system
allows to test, is illustrated by its capacity to promote phage
uptake in the T7 select system. This system favors rapid uptake
exclusively through high affinity binding to endothelial cells,
since it allows the surface expression of less than one peptide
copy per phage. In certain embodiments, Endo5 is the first CPP
engineered specifically for high endothelial cell internalization
though the competitive selection approach.
[0077] Without wishing to be limited by any theory, the
intracellular distribution of cargos towards their target may be at
least partially, independent of the CPP sequence based on the fact
that Cav fused to two completely different CPPs (Endo5 or AP)
attenuated eNOS activity, inhibited vascular permeability and
co-localized after a two-hour "chase." Further, it is possible that
Endo5, as well as other CPP, may allow various exit pathways from
internalization organelles and/or direct the intracellular
localization of cargo molecules differently. However, the
difference in uptake rate between Endo5 and AP is the likely
mechanism to rationalize the difference in potency between
Endo5-Cav and AP-Cav. This is also supported by the data
documenting the similar localization of Endo5 and AP in live cells.
On the other hand, the increase in Cav potency when fused to Endo5
and the near saturation of AP-Cav effect on eNOS activity at high
doses suggest that AP-Cav effect might be limited by an overlapping
rate of internalization and elimination/degradation rather than by
a limitation of the pharmacophore (Cav). Without wishing to be
limited by any theory, this highlights the interest in identifying
highly potent CPP sequences, such as sequences comprising
Endo5.
Compositions
[0078] The invention includes an isolated transport peptide that
crosses a cell membrane. In certain embodiments, the peptide, or a
salt or solvate thereof, comprises the amino acid sequence RRPPR
(SEQ ID NO: 1). In other embodiments, the transport peptide, or a
salt or solvate thereof, consists essentially of SEQ ID NO: 1. In
yet other embodiments, the transport peptide, or a salt or solvate
thereof, consists of SEQ ID NO: 1. In yet other embodiments, the
transport peptide binds to a target cell or crosses a cell
membrane. In yet other embodiments, the cell comprises an
endothelial cell, cardiac cell, immune cell, skeletal muscle cell
or brain cell. In yet other embodiments, the cell consists of an
endothelial cell, cardiac cell, immune cell, skeletal muscle cell
or brain cell.
[0079] The invention further provides a pharmaceutical composition
comprising a transport peptide comprising SEQ ID NO: 1 and a
pharmaceutically acceptable carrier.
[0080] In certain embodiments, the compositions of the invention
further comprise a pharmaceutically acceptable carrier.
[0081] In certain embodiments, the transport construct comprises a
cargo moiety linked to a transport peptide comprising SEQ ID NO: 1.
In other embodiments, the transport peptide consists of SEQ ID NO:
1. In yet other embodiments, the cargo moiety is at least one
selected from the group consisting of a nucleic acid (and analogues
thereof, such as a peptide nucleic acid or "PNA"); peptide;
protein; oligosaccharide; lipid; glycolipid; lipoprotein;
therapeutic drug; UV-vis, fluorescent or radioactive label; imaging
agent; diagnostic agent; prophylactic agent; liposome and virus
(such as T-7 bacteriophage).
[0082] In certain embodiments, the cargo moiety is at least one
selected from the group consisting of SEQ ID NOs: 3-6. In other
embodiments, the transport construct comprises at least one
sequence selected from the group consisting of SEQ ID NO: 1-SEQ ID
NO: 3; SEQ ID NO: 1-SEQ ID NO: 4; SEQ ID NO: 1-SEQ ID NO: 5; SEQ ID
NO: 1-SEQ ID NO: 6; SEQ ID NO: 3-SEQ ID NO: 1; SEQ ID NO: 4-SEQ ID
NO: 1; SEQ ID NO: 5-SEQ ID NO: 1; and SEQ ID NO: 6-SEQ ID NO: 1. In
yet other embodiments, the transport construct is selected from the
group consisting of SEQ ID NO: 1-SEQ ID NO: 3; SEQ ID NO: 1-SEQ ID
NO: 4; SEQ ID NO: 1-SEQ ID NO: 5; SEQ ID NO: 1-SEQ ID NO: 6; SEQ ID
NO: 3-SEQ ID NO: 1; SEQ ID NO: 4-SEQ ID NO: 1; SEQ ID NO: 5-SEQ ID
NO: 1; and SEQ ID NO: 6-SEQ ID NO: 1.
[0083] The cargo moiety may be combined with or linked to the
transport peptide to form the transport construct of the present
invention. The transport peptide and the cargo moiety are combined
or linked in such a manner that they remain combined or linked
under the conditions in which the transport construct is used
(e.g., under conditions in which the transport construct is
administered to an individual). In certain embodiments, the cargo
moiety is covalently linked to the transport peptide through a
linker or a chemical bond. In other embodiments, the linker
comprises a disulfide bond, or the chemical bond between the cargo
moiety and the transport peptide comprises a disulfide bond. In yet
other embodiments, the cargo moiety comprises a peptide moiety. In
yet other embodiments, the transport peptide is covalently linked
through an amide bond to the N-terminus of the peptide moiety of
the cargo moiety. In yet other embodiments, the transport peptide
is covalently linked through an amide bond to the C-terminus of the
peptide moiety of the cargo moiety. In yet other embodiments, the
transport peptide is covalently linked through an amide bond to the
N-terminus and the C-terminus of the peptide moiety of the cargo
moiety. Alternatively, the transport peptide and the cargo moiety
are combined through a noncovalent linkage, such as electrostatic
and/or hydrophobic interaction.
[0084] The invention includes functionally equivalent variants of
peptides described elsewhere herein. Such variants include peptides
with amino acid substitutions that maintain the functional
integrity of the original peptide. Examples of amino acid
substitutions include those that result in changes to the peptide
wherein similar charge, polarity, hydrophobicity or structure of
the original amino acid is maintained. Peptide variants also
include peptide mimetics. Peptide mimetics include chemically
modified peptides and peptide-like molecules containing
non-naturally occurring amino acids.
[0085] In certain embodiments, the peptides of the present
invention may be obtained from sources in which they occur in
nature or produced using known techniques, such as chemical
synthesis or genetic engineering methods (e.g., recombinant DNA or
RNA technology). In other embodiments, the peptides of the
invention may be prepared using standard solid phase (or solution
phase) peptide synthesis methods, as is known in the art. In
addition, the DNA encoding these peptides may be synthesized using
commercially available oligonucleotide synthesis instrumentation
and produced recombinantly using standard recombinant production
systems.
[0086] In certain embodiments, isolated peptides of the present
invention are relatively free from unrelated peptides, as well as
contaminating polypeptides, lipids, nucleic acids and other
cellular material that normally are associated with the peptide in
a cell or that are associated with the peptide in a library.
[0087] The transport constructs of the invention are useful for the
delivery of cargo moieties across the cell membrane. The transport
constructs of the invention are also useful for the delivery of
cargo moieties to a target cell (e.g., a specific cell type, such
as but not limited to a cardiac cell, an endothelial cell, or an
immune cell) and for the delivery of cargo moieties to a target
cell and/or across the membrane of the target cell.
[0088] The transport peptides of the present invention have the
ability to cross the cell membrane of a cell (e.g., internalize
into the cell). For example, in certain embodiments of the
invention, a transport peptide translocates from the extracellular
environment of a cell, penetrates the lipid bilayer of the cell
membrane, and crosses the cell membrane into the intracellular
environment of the cell. In other embodiments, the transport
peptides of the present invention bind to a target cell. In yet
other embodiments, the transport peptides bind to and cross the
cell membrane of a target cell. A target cell is a specific cell
type such as, for example, a cardiac cell, an immune cell, a skin
cell (e.g., an endothelial cell), a skeletal muscle cell or a brain
cell (e.g. a neuron) but may be any cell, including human and
nonhuman cells.
[0089] In a non-limiting example, a transport peptide of the
invention is linked to a cargo moiety and transports the cargo
moiety across the cell membrane of a cell. For example, In certain
embodiments, a protein, such as caveolin or a transcription factor,
linked to a transport peptide is carried from the extracellular
environment of a cell and transported across the cell membrane and
into the intracellular environment of the cell. In other
embodiments, a transport peptide of the invention linked to a cargo
moiety binds the cargo moiety to a target cell (e.g., a cardiac
cell). In yet other embodiments, the transport peptide linked to a
cargo moiety binds the cargo moiety to a target cell (e.g., a
cardiac cell), and transports the cargo moiety from the
extracellular environment of the target cell across the cell
membrane and into the intracellular environment of the target
cell.
[0090] In certain embodiments, the cargo moiety comprises an
organic or inorganic compound. The organic compound may be isolated
from nature (e.g., from cells in which it occurs) or may be
produced using known methods, such as genetic engineering methods
(e.g., recombinant DNA or RNA technology) or chemical synthetic
methods. For example, an organic molecule may be an RNA molecule,
polypeptide or a fragment thereof, which may be isolated from a
cell, expressed from a recombinant nucleic acid molecule or
synthesized chemically. An organic molecule also can be a
non-naturally occurring molecule. A non-limiting example of a
non-naturally occurring molecule is a nucleic acid sequence
containing non-naturally occurring nucleoside analogs or
phosphorothioate bonds that link the nucleotides and protect
against degradation by nucleases. A ribonucleotide containing a
2-methyl group, instead of the normal hydroxyl group, bonded to the
2'-carbon atom of ribose residues, is an example of a non-naturally
occurring RNA molecule that is resistant to enzymatic and chemical
degradation. Other examples of non-naturally occurring organic
molecules include RNA containing 2'-aminopyrimidines (wherein such
RNA is 1,000 times more stable in human serum and urine as compared
to naturally occurring RNA; Lin et al., 1994, Nucl. Acids Res.
22:5229-5234, and Jellinek et al., 1995, Biochemistry,
34:11363-11372).
[0091] In certain embodiments, the cargo moiety comprises a DNA, a
RNA or a nucleic acid analog. The DNA or RNA may be an
oligo(deoxy)nucleotide of any length. Such nucleic acid molecules
may be linear, circular or supercoiled; may be single-stranded or
double-stranded DNA or RNA; or may be a DNA/RNA hybrid. Nucleic
acid analogs include charged and uncharged backbone analogs, such
as phosphonates (e.g., methyl phosphonates), phosphoramidates (NY
or N5'), thiophosphates, uncharged morpholino-based polymers, and
peptide nucleic acids (PNAs). Such molecules may be used in a
variety of therapeutic regimens, including enzyme replacement
therapy, gene therapy and anti-sense therapy, for example. Peptide
nucleic acids (PNAs) are analogs of DNA. The backbone of a PNA is
formed by peptide bonds rather than phosphate esters, making it
well-suited for anti-sense applications. Since the backbone is
uncharged, PNA/DNA or PNA/RNA duplexes exhibit greater than normal
thermal stability. PNAs have the additional advantage that they are
not recognized by nucleases or proteases. PNAs may be synthesized
on an automated peptides synthesizer using standard t-Boc
chemistry. The PNA may be linked to a transport peptide of the
invention using known methods in the art.
[0092] In certain embodiments, the cargo moiety is a polypeptide.
In other embodiments, the cargo moiety comprises caveolin or a
fragment thereof. In yet other embodiments, the cargo moiety is a
transcription factor or a nuclear localization peptide. In yet
other embodiments, two cargo moieties, one comprising a
transcription factor and the other comprising a nuclear
localization peptide, are present in the transport construct of the
invention.
[0093] In certain embodiments, the cargo moiety comprises a label,
such as a dye or a radioactively labeled compound. In other
embodiments, the cargo moiety comprises rhodamine. In yet other
embodiments, the cargo moiety comprises a marker, such as green
fluorescent protein, blue fluorescent protein, yellow fluorescent
protein, biotin or mixtures thereof.
[0094] In a non-limiting example, recombinant techniques may be
used to covalently attach a transport peptide to a cargo moiety,
such as joining DNA or RNA coding for the transport peptide with
DNA or RNA coding for the cargo moiety and expressing the encoded
products in an appropriate host cell (a cell capable of expressing
the transport construct). Alternatively, the two separate
nucleotide sequences may be expressed in a cell or can be
synthesized chemically and subsequently combined, using known
techniques. Alternatively, the transport peptide-cargo moiety may
be synthesized chemically as a single amino acid sequence and,
thus, combining them is not needed.
[0095] In certain embodiments, when there is more than one cargo
moiety linked to the transport peptide, the more than one moiety
may be the same or different. In other embodiments, the cargo
moiety or moieties are linked to the transport peptide at either
the N- or C-terminus of the transport peptide. In the case wherein
there are at least two cargo moieties linked to the transport
peptide, one cargo moiety may be linked at the N-terminus of the
transport peptide and one cargo moiety may be linked at the
C-terminus of the transport peptide. Alternatively, more than one
cargo moiety may be linked to either the N- or C-terminus of the
transport peptide.
[0096] In certain embodiments, the cargo moiety may be linked to a
transport peptide of the present invention either directly (I.e.,
through a chemical bond) or indirectly by means of a linker.
Linkers include, for example, one or more amino acid residues. The
linker may be, for example, a short sequence of 10 amino acid
residues (e.g., 1 to 10, 1 to 5 or 1 to 4 amino acid residues), and
may optionally include a cysteine residue through which the linker
binds to the transport peptide or cargo moiety of the transport
construct. A linker may also be a group such as a sulfhydryl group
or carboxyl group. Suitable linkers include bi- and
multi-functional alkyl, aryl, aralkyl or peptidic moieties, alkyl,
aryl or aralkyl aldehydes, acids, esters and anhydrides, sulfydryl
or carboxyl groups, such as maleimido benzoic acid derivatives,
maleimido proprionic acid derivatives and succinimido derivatives,
or may be derived from cyanuric bromide or chloride,
carbonyldiimidazole, succinimidyl esters or sulfonic halides. The
functional groups on the linker used to form covalent bonds between
linker and cargo moiety on the one hand, as well as linker and
transport peptide on the other hand, may be two or more of e.g.,
amino, hydrazine, hydroxyl, thiol, maleimido, carbonyl, and
carboxyl groups.
[0097] In certain embodiments, the transport construct may
dissociate in vitro or in vivo into the cargo moiety and transport
peptide by way of chemical or enzymatic cleavage. In other
embodiments, the linker comprises amino acid residues, and the in
vitro or in vivo cleavage occurs within the linker.
[0098] In certain embodiments, wherein the cargo moiety is a
polypeptide, the cargo moiety is linked to the transport peptide as
a fusion protein by means of recombinant technology. A fusion
protein is the co-linear, covalent linkage of two or more proteins
via their polypeptide backbones, through genetic expression of a
nucleic acid molecule encoding those proteins. The nucleic acid
encoding the cargo moiety of the fusion protein is in-frame with
the nucleic acid encoding the transport peptide. "In-frame"
indicates that the nucleic acid sequence encoding the cargo moiety
is in the correct reading frame as the nucleic acid sequence
encoding the transport peptide. Therefore, the correct amino acid
sequences is translated for both the transport peptide and cargo
moiety of the fusion protein.
[0099] In certain embodiments, the cargo moiety is conjugated to
the transport peptide via chemical cross-linking. Numerous chemical
cross-linking methods are known and useful for linking the
transport peptides of this invention to a cargo moiety. Coupling of
the cargo moiety and the transport peptide may be accomplished via
a coupling or linking agent. Intermolecular cross-linking reagents
that may be utilized are exemplified in Means & Feeney,
Chemical Modification of Proteins, Holden-Day, 1974, pp. 39-43, and
Wong, Chemistry of Protein Conjugation and Cross-Linking, CRC Press
(1991). Among these reagents are, for example, N-succinimidyl
3-(2-pyridyldithio) propionate ("SPDP") or N,N'-(1,3-phenylene)
bismaleimide (both of which are highly specific for sulfydryl
groups and form irreversible linkages);
N,N'-ethylene-bis-(iodoacetamide) or other such reagent having 6 to
11 carbon methylene bridges (which are relatively specific for
sulfhydryl groups); and 1,5-difluoro-2,4-dinitrobenzene (which
forms irreversible linkages with amino and tyrosine groups). Other
cross-linking reagents useful for this purpose include:
p,p'-difluoro-m,m'-dinitrodiphenylsulfone (which forms irreversible
cross-linkages with amino and phenolic groups); dimethyl
adipimidate (which is specific for amino groups);
phenol-1,4-disulfonylchloride (which reacts principally with amino
groups); hexamethylenediisocyanate or diisothiocyanate, or
azophenyl-p-diisocyanate (which reacts mainly with amino groups);
glutaraldehyde (which reacts with different side chains) and
disdiazobenzidine (which reacts primarily with tyrosine and
histidine).
[0100] In certain embodiments, the cross-linking reagents yields a
transport construct that is essentially non-cleavable under
cellular conditions. In other embodiments, the cross-linking
reagent contains a covalent bond, such as a disulfide, that is
cleavable under cellular conditions. For example,
dithiobis(succinimidylpropionate) ("DSP"), Traut's reagent and
N-succinimidyl 3-(2-pyridyldithio) propionate ("SPDP") are
well-known cleavable cross-linkers. The use of a cleavable
cross-linking reagent permits the transport peptide to separate
from the cargo moiety after delivery into the target cell. A
construct comprising a direct disulfide linkage may also be useful
within the methods of the invention. In certain embodiments, the
cargo moiety is covalently linked to the transport peptide through
a linker or a chemical bond. In other embodiments, the
cross-linking reagent such as
N-gamma-maleimidobutyryloxy-succinimide ester ("GMBS") and
sulfo-GMBS have reduced immunogenicity.
[0101] The present invention further includes a composition
comprising an isolated nucleic acid molecule that encodes the
polypeptide having the fusion peptides and conservative nucleotide
substitutions thereof, in certain embodiments in isolated form to
generate the compositions of the invention. Conservative nucleotide
substitutions include nucleotide substitutions that do not affect
the coding for a particular amino acid as most amino acids have
more than one codon. Conservative nucleotide substitutions thus
also include silent mutations and differential codon usage.
[0102] In certain embodiments, the nucleic acid encodes a transport
peptide comprising SEQ ID NO: 1. In other embodiments, the nucleic
acid encodes a transport peptide consisting of SEQ ID NO: 1. In
other embodiments, the nucleic acid comprises 5'-CGGCGCCCGCCTCGT-3'
(SEQ ID NO: 7).
[0103] In certain embodiments, the composition further comprises a
nucleic acid encoding at least one cargo moiety. In other
embodiments, the cargo moiety is selected from the group consisting
of a peptide; a protein; a biologically active compound; a label;
an imaging agent; a diagnostic agent; a therapeutic agent; and a
prophylactic agent. In yet other embodiments, the cargo moiety
comprises at least one selected from the group consisting of SEQ ID
NOs: 3-6. In yet other embodiments, the composition further
comprises a pharmaceutically acceptable carrier.
[0104] The invention further includes an expression vector and an
isolated host cell comprising nucleic acid encoding a peptide
comprising SEQ ID NO: 1. In certain embodiments, the peptide
consists of SEQ ID NO: 1.
[0105] The invention also includes an expression vector and an
isolated host cell comprising nucleic acid encoding a cargo moiety
linked to the peptide comprising SEQ ID NO: 1. In certain
embodiments, the peptide consists of SEQ ID NO: 1.
[0106] In certain embodiments, the transport construct comprises a
fusion protein. In other embodiments, the vector or host cell
further comprises transcriptional activation elements that allow
for the expression of the nucleic acid encoding the transport
peptide. Expression system vectors, which incorporate the necessary
regulatory elements for protein expression, as well as restriction
endonuclease sites that facilitate cloning of the desired sequences
into the vector, are known to those skilled in the art. In certain
embodiments, the cargo moiety is in-frame with the nucleic acid
encoding the transport peptide.
[0107] In a non-limiting example, a recombinant DNA expression
vector containing the elements previously described is introduced
into an appropriate host cell (i.e., a cell capable of expressing
the transport construct) where cellular mechanisms of the host cell
direct the expression of the fusion protein encoded by the
recombinant DNA expression vector. Alternately, cell-free systems
known to those skilled in the art may be used for expression of the
fusion protein.
[0108] The purified fusion protein produced by the expression
vector host cell system may then be administered to the target
cell, where the transport peptide mediates the import of the fusion
protein through the cell membrane of the target cell into the
interior of the cell. A target cell is a specific cell type such
as, for example, a cardiac cell, an immune cell, a skin cell, such
as an epithelial cell; a skeletal muscle cell or a brain cell
(e.g., a neuron), but may be any cell, including human and nonhuman
cells.
[0109] An expression vector host cell system may be selected from
among a number of such systems known to those skilled in the art.
In certain embodiments, the fusion protein may be expressed in
isolated host cells, such as Escherichia coli. In other
embodiments, fusion proteins may be expressed in other bacterial
expression systems, viral expression systems, eukaryotic expression
systems, or cell-free expression systems. Cellular hosts used by
those skilled in the art include, but are not limited to, isolated
host cells such as, for example, Bacillus subtilis, yeast such as
Saccharomyces cerevisiae, Saccharomyces carlsbergenesis,
Saccharomyces pombe, and Pichia pastoris, as well as mammalian
cells such as NIH3T3, HeLa, HEK293, HUVEC, rat aortic smooth muscle
cells and adult human smooth muscle cells. The expression vector
selected by one skilled in the art includes transcriptional
activation elements such as promoter elements and other regulatory
elements appropriate for the host cell or cell-free system in which
the fusion protein will be expressed. In mammalian expression
systems, for example, suitable expression vectors may include DNA
plasmids, DNA viruses, and RNA viruses. In bacterial expression
systems, suitable vectors may include plasmid DNA and bacteriophage
vectors.
[0110] Examples of specific expression vector systems include the
pBAD/gIII vector (Invitrogen, Carlsbad, Calif.) system for protein
expression in E. coli, which is regulated by the transcriptional
regulator AraC. An example of a vector for mammalian expression is
the pcDNA3.1N5-His-TOPO eukaryotic expression vector (Invitrogen).
In this vector, the transport construct maybe expressed at high
levels under the control of a strong cytomegalovirus (CMV)
promoter. A C-terminal polyhistidine (His.sub.6) tag enables
transport construct purification using nickel-chelating resin.
Secreted protein produced by this vector may be detected using an
anti-His (C-term) antibody.
[0111] A baculovirus expression system may also be used for
production of a transport construct comprising the transport
peptide and a cargo moiety wherein the cargo moiety is a
polypeptide. A commonly used baculovirus is AcMNPV. Cloning of the
transport construct DNA may be accomplished by using homologous
recombination. In a non-limiting example, the transport construct
DNA sequence is cloned into a transfer vector containing a
baculovirus promoter flanked by baculovirus DNA, particularly DNA
from the polyhedrin gene. This DNA is transfected into insect
cells, where homologous recombination occurs to insert the
transport construct DNA into the genome of the parent virus.
Recombinants are identified by altered plaque morphology.
[0112] Many transport constructs in which the cargo moiety is a
peptide or protein that may not be appropriately
post-translationally modified in bacterial expression systems may
instead be expressed with baculovirus vectors. Enzymes, signaling
molecules, mediators of cell cycle control, transcription factors,
antigenic peptides, full-length protein products of viral,
bacterial, or other origin for use in vaccine therapy, protein
products of human cells for use in cancer vaccine therapy, toxins,
and proteins involved in intracellular signaling systems that may
not be appropriately post-translationally modified in bacterial
expression systems may be expressed with baculovirus vectors.
[0113] Proteins as described above may also be produced by the
method of the present invention by mammalian viral expression
systems. An ecdysone-inducible mammalian expression system
(Invitrogen, Carlsbad, Calif.) may also be used to express the
transport construct wherein the transport construct is a fusion
protein.
[0114] In certain embodiments, yeast host cells, such as Pichia
pastoris, may be used for the production of a transport construct
by the method of the present invention. Expression of heterologous
proteins from plasmids transformed into Pichia has been described
by U.S. Pat. No. 5,002,876 to Sreekrishna et al. Vectors for
expression in Pichia of a fusion protein comprising a transport
peptide of the present invention and a cargo moiety wherein the
cargo moiety is a peptide or protein are commercially available as
part of a Pichia Expression Kit (Invitrogen, Carlsbad, Calif.).
[0115] Purification of heterologous protein produced in Pichia was
described by U.S. Pat. No. 5,004,688 to Craig et al., and
techniques for protein purification from yeast expression systems
are well known to those skilled in the art. In the Pichia system,
commercially available vectors may be selected from among those
that are more suited for the production of cytosolic,
non-glycosylated proteins and those that are more suited for the
production of secreted, glycosylated proteins, or those directed to
an intracellular organelle, so that appropriate protein expression
may be optimized for the cargo moiety of choice that is a
polypeptide.
Methods
[0116] The invention includes a method of delivering a cargo moiety
to or into, also referred to as (in)to, a target cell. In certain
embodiments, the method comprises contacting the target cell with a
transport construct, wherein the transport construct comprises a
cargo moiety and a transport peptide comprising SEQ ID NO: 1,
whereby the cargo moiety is delivered (in)to the target cell.
[0117] The invention further includes a method of delivering a
cargo moiety (in)to a target cell of a subject in need thereof. In
certain embodiments, the method comprises administering to the
subject a therapeutically effective amount of a pharmaceutically
acceptable composition comprising a transport construct, wherein
the transport construct comprises the cargo moiety and a transport
peptide comprising SEQ ID NO: 1, whereby the cargo moiety is
delivered (in)to the target cell of the subject.
[0118] In certain embodiments, the cargo moiety is covalently
linked to the transport peptide through a linker or a chemical
bond. In other embodiments, the linker comprises a disulfide bond,
or the chemical bond between the cargo moiety and the transport
peptide comprises a disulfide bond. In yet other embodiments, the
cargo moiety comprises a peptide moiety. In yet other embodiments,
the transport peptide is covalently linked through an amide bond to
the N-terminus of the peptide moiety of the cargo moiety. In yet
other embodiments, the transport peptide is covalently linked
through an amide bond to the C-terminus of the peptide moiety of
the cargo moiety. In yet other embodiments, the transport peptide
is covalently linked through an amide bond to the N-terminus and
the C-terminus of the peptide moiety of the cargo moiety.
[0119] In certain embodiments, the transport peptide consists of
SEQ ID NO: 1. In other embodiments, the cargo moiety is at least
one selected from the group consisting of a nucleic acid; a
peptide; a protein; an oligosaccharide; a lipid; a glycolipid; a
lipoprotein; a small molecule compound; a therapeutic drug; an
UV-vis, fluorescent or radioactive label; an imaging agent; a
diagnostic agent; a prophylactic agent; a liposome and a virus. In
yet other embodiments, the target cell comprises an endothelial
cell, a cardiac cell, an immune cell, a skeletal muscle cell or a
brain cell. In yet other embodiments, a composition of the
invention is administered to the subject by at least one route
selected from the group consisting of oral, transmucosal, topical,
transdermal, intradermal, subcutaneous, ophthalmic, intravitreal,
subconjunctival, suprachoroidal, intracameral, inhalational,
intrabronchial, pulmonary, intravenous, intra-arterial,
intraduodenal, intravesical, parenteral, intrathecal, intramuscular
and intragastrical. In yet other embodiments, the subject is a
mammal. In yet other embodiments, the mammal is human.
Combination Therapies
[0120] The compositions useful within the present invention are
intended to be useful in the methods of present invention in
combination with one or more additional compounds useful for
treating the diseases or disorders contemplated within the
invention. These additional compounds may comprise compounds of the
present invention or compounds, e.g., commercially available
compounds, known to treat, prevent, or reduce the symptoms of the
diseases or disorders contemplated within the invention.
[0121] A synergistic effect may be calculated, for example, using
suitable methods such as, for example, the Sigmoid-E.sub.max
equation (Holford & Scheiner, 19981, Clin. Pharmacokinet. 6:
429-453), the equation of Loewe additivity (Loewe & Muischnek,
1926, Arch. Exp. Pathol Pharmacol. 114: 313-326) and the
median-effect equation (Chou & Talalay, 1984, Adv. Enzyme
Regul. 22: 27-55). Each equation referred to above may be applied
to experimental data to generate a corresponding graph to aid in
assessing the effects of the drug combination. The corresponding
graphs associated with the equations referred to above are the
concentration-effect curve, isobologram curve and combination index
curve, respectively.
Administration/Dosage/Formulations
[0122] The regimen of administration may affect what constitutes an
effective amount. The therapeutic formulations may be administered
to the patient either prior to or after the onset of a disease or
disorder. Further, several divided dosages, as well as staggered
dosages may be administered daily or sequentially, or the dose may
be continuously infused, or may be a bolus injection. Further, the
dosages of the therapeutic formulations may be proportionally
increased or decreased as indicated by the exigencies of the
therapeutic or prophylactic situation.
[0123] Administration of the compositions useful within the present
invention to a patient, in certain embodiments a mammal, in other
embodiments a human, may be carried out using known procedures, at
dosages and for periods of time effective to treat a disease or
disorder in the patient. An effective amount of the therapeutic
compound necessary to achieve a therapeutic effect may vary
according to factors such as the state of the disease or disorder
in the patient; the age, sex, and weight of the patient; and the
ability of the therapeutic compound to treat a disease or disorder
in the patient. Dosage regimens may be adjusted to provide the
optimum therapeutic response. For example, several divided doses
may be administered daily or the dose may be proportionally reduced
as indicated by the exigencies of the therapeutic situation. A
non-limiting example of an effective dose range for a therapeutic
compound of the invention is from about 1 and 5,000 mg/kg of body
weight/per day. One of ordinary skill in the art would be able to
study the relevant factors and make the determination regarding the
effective amount of the therapeutic compound without undue
experimentation.
[0124] Actual dosage levels of the active ingredients in the
pharmaceutical compositions of this invention may be varied so as
to obtain an amount of the active ingredient that is effective to
achieve the desired therapeutic response for a particular patient,
composition, and mode of administration, without being toxic to the
patient.
[0125] In particular, the selected dosage level depends upon a
variety of factors including the activity of the particular
compound employed, the time of administration, the rate of
excretion of the compound, the duration of the treatment, other
drugs, compounds or materials used in combination with the
compound, the age, sex, weight, condition, general health and prior
medical history of the patient being treated, and like factors
well, known in the medical arts.
[0126] A medical doctor, e.g., physician or veterinarian, having
ordinary skill in the art may readily determine and prescribe the
effective amount of the pharmaceutical composition required. For
example, the physician or veterinarian could start doses of the
compounds of the invention employed in the pharmaceutical
composition at levels lower than that required in order to achieve
the desired therapeutic effect and gradually increase the dosage
until the desired effect is achieved.
[0127] In particular embodiments, it is especially advantageous to
formulate the compound in dosage unit form for ease of
administration and uniformity of dosage. Dosage unit form as used
herein refers to physically discrete units suited as unitary
dosages for the patients to be treated; each unit containing a
predetermined quantity of therapeutic compound calculated to
produce the desired therapeutic effect in association with the
required pharmaceutical vehicle. The dosage unit forms of the
invention are dictated by and directly dependent on (a) the unique
characteristics of the therapeutic compound and the particular
therapeutic effect to be achieved, and (b) the limitations inherent
in the art of compounding/formulating such a therapeutic compound
for the treatment of a disease or disorder in a patient.
[0128] In certain embodiments, the compositions useful within the
invention are formulated using one or more pharmaceutically
acceptable excipients or carriers. In certain embodiments, the
pharmaceutical compositions of the invention comprise a
therapeutically effective amount of a compound useful within the
invention and a pharmaceutically acceptable carrier.
[0129] The carrier may be a solvent or dispersion medium
containing, for example, water, ethanol, polyol (for example,
glycerol, propylene glycol, and liquid polyethylene glycol, and the
like), suitable mixtures thereof, and vegetable oils. The proper
fluidity may be maintained, for example, by the use of a coating
such as lecithin, by the maintenance of the required particle size
in the case of dispersion and by the use of surfactants. Prevention
of the action of microorganisms may be achieved by various
antibacterial and antifungal agents, for example, parabens,
chlorobutanol, phenol, ascorbic acid, and thimerosal. In many
cases, isotonic agents, for example, sugars, sodium chloride, or
polyalcohols such as mannitol and sorbitol, can be included in the
composition. Prolonged absorption of the injectable compositions
may be brought about by including in the composition an agent which
delays absorption, for example, aluminum monostearate or
gelatin.
[0130] In certain embodiments, the compositions useful within the
invention are administered to the patient in dosages that range
from one to five times per day or more. In other embodiments, the
compositions useful within the invention are administered to the
patient in range of dosages that include, but are not limited to,
once every day, every two, days, every three days to once a week,
and once every two weeks. It will be readily apparent to one
skilled in the art that the frequency of administration of the
various combination compositions useful within the invention will
vary from individual to individual depending on many factors
including, but not limited to, age, disease or disorder to be
treated, gender, overall health, and other factors. Thus, the
invention should not be construed to be limited to any particular
dosage regime and the precise dosage and composition to be
administered to any patient will be determined by the attending
physical taking all other factors about the patient into
account.
[0131] Compounds for administration may be in the range of from
about 1 .mu.g to about 10,000 mg, about 20 .mu.g to about 9,500 mg,
about 40 .mu.g to about 9,000 mg, about 75 .mu.g to about 8,500 mg,
about 150 .mu.g to about 7,500 mg, about 200 .mu.g to about 7,000
mg, about 3050 .mu.g to about 6,000 mg, about 500 .mu.g to about
5,000 mg, about 750 .mu.g to about 4,000 mg, about 1 mg to about
3,000 mg, about 10 mg to about 2,500 mg, about 20 mg to about 2,000
mg, about 25 mg to about 1,500 mg, about 50 mg to about 1,000 mg,
about 75 mg to about 900 mg, about 100 mg to about 800 mg, about
250 mg to about 750 mg, about 300 mg to about 600 mg, about 400 mg
to about 500 mg, and any and all whole or partial increments
therebetween.
[0132] In certain embodiments, the dose of a compound is from about
1 mg and about 2,500 mg. In other embodiments, a dose of a compound
of the invention used in compositions described herein is less than
about 10,000 mg, or less than about 8,000 mg, or less than about
6,000 mg, or less than about 5,000 mg, or less than about 3,000 mg,
or less than about 2,000 mg, or less than about 1,000 mg, or less
than about 500 mg, or less than about 200 mg, or less than about 50
mg. Similarly, in certain embodiments, a dose of a second compound
(i.e., a drug used for treating a disease or disorder) as described
herein is less than about 1,000 mg, or less than about 800 mg, or
less than about 600 mg, or less than about 500 mg, or less than
about 400 mg, or less than about 300 mg, or less than about 200 mg,
or less than about 100 mg, or less than about 50 mg, or less than
about 40 mg, or less than about 30 mg, or less than about 25 mg, or
less than about 20 mg, or less than about 15 mg, or less than about
10 mg, or less than about 5 mg, or less than about 2 mg, or less
than about 1 mg, or less than about 0.5 mg, and any and all whole
or partial increments thereof.
[0133] In certain embodiments, the present invention is directed to
a packaged pharmaceutical composition comprising a container
holding a therapeutically effective amount of a compound of the
invention, alone or in combination with a second pharmaceutical
agent; and instructions for using the compound to treat, prevent,
or reduce one or more symptoms of a disease or disorder in a
patient.
[0134] Formulations may be employed in admixtures with conventional
excipients, i.e., pharmaceutically acceptable organic or inorganic
carrier substances suitable for oral, parenteral, nasal,
intravenous, subcutaneous, enteral, or any other suitable mode of
administration, known to the art. The pharmaceutical preparations
may be sterilized and if desired mixed with auxiliary agents, e.g.,
lubricants, preservatives, stabilizers, wetting agents,
emulsifiers, salts for influencing osmotic pressure buffers,
coloring, flavoring and/or aromatic substances and the like. They
may also be combined where desired with other active agents, e.g.,
other cognition improving agents.
[0135] The term "container" includes any receptacle for holding the
pharmaceutical composition. For example, In certain embodiments,
the container is the packaging that contains the pharmaceutical
composition. In other embodiments, the container is not the
packaging that contains the pharmaceutical composition, i.e., the
container is a receptacle, such as a box or vial that contains the
packaged pharmaceutical composition or unpackaged pharmaceutical
composition and the instructions for use of the pharmaceutical
composition. Moreover, packaging techniques are well known in the
art. It should be understood that the instructions for use of the
pharmaceutical composition may be contained on the packaging
containing the pharmaceutical composition, and as such the
instructions form an increased functional relationship to the
packaged product. However, it should be understood that the
instructions may contain information pertaining to the compound's
ability to perform its intended function, e.g., treating,
preventing, or reducing a disease or disorder in a patient.
[0136] Routes of administration of any of the compositions of the
invention include oral, buccal, topical, transdermal, intradermal,
subcutaneous, transmucosal [e.g., sublingual, lingual,
(trans)buccal, (trans)urethral, vaginal (e.g., trans- and
perivaginally), (intra)nasal and (trans)rectall, ophthalmic (e.g.,
intravitreal, subconjunctival, suprachoroidal, intracameral),
inhalational, intrabronchial, pulmonary, intraduodenal,
intravenous, intra-arterial, intravesical, parenteral, intrathecal,
intramuscular or intragastrical route. The compounds for use in the
invention may be formulated for administration by any suitable
route considered herein.
[0137] Suitable compositions and dosage forms include, for example,
tablets, capsules, caplets, pills, gel caps, troches, dispersions,
suspensions, solutions, syrups, granules, beads, transdermal
patches, gels, powders, pellets, magmas, lozenges, creams, pastes,
plasters, lotions, discs, suppositories, liquid sprays for nasal or
oral administration, dry powder or aerosolized formulations for
inhalation, compositions and formulations for intravesical
administration and the like. It should be understood that the
formulations and compositions that would be useful in the present
invention are not limited to the particular formulations and
compositions that are described herein.
Oral Administration
[0138] For oral application, particularly suitable are tablets,
dragees, liquids, drops, suppositories, or capsules, caplets and
gelcaps. The compositions intended for oral use may be prepared
according to any method known in the art and such compositions may
contain one or more agents selected from the group consisting of
inert, non-toxic pharmaceutically excipients which are suitable for
the manufacture of tablets. Such excipients include, for example an
inert diluent such as lactose; granulating and disintegrating
agents such as cornstarch; binding agents such as starch; and
lubricating agents such as magnesium stearate. The tablets may be
uncoated or they may be coated by known techniques for elegance or
to delay the release of the active ingredients. Formulations for
oral use may also be presented as hard gelatin capsules wherein the
active ingredient is mixed with an inert diluent.
[0139] For oral administration, the compounds may be in the form of
tablets or capsules prepared by conventional means with
pharmaceutically acceptable excipients such as binding agents
(e.g., polyvinylpyrrolidone, hydroxypropylcellulose or
hydroxypropylmethylcellulose); fillers (e.g., cornstarch, lactose,
microcrystalline cellulose or calcium phosphate); lubricants (e.g.,
magnesium stearate, talc, or silica); disintegrates (e.g., sodium
starch glycollate); or wetting agents (e.g., sodium lauryl
sulphate). If desired, the tablets may be coated using suitable
methods and coating materials such as OPADRY.TM. film coating
systems available from Colorcon, West Point, Pa. (e.g., OPADRY.TM.
OY Type, OYC Type, Organic Enteric OY-P Type, Aqueous Enteric OY-A
Type, OY-PM Type and OPADRY.TM. White, 32K18400). Liquid
preparation for oral administration may be in the form of
solutions, syrups or suspensions. The liquid preparations may be
prepared by conventional means with pharmaceutically acceptable
additives such as suspending agents (e.g., sorbitol syrup, methyl
cellulose or hydrogenated edible fats); emulsifying agent (e.g.,
lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily
esters or ethyl alcohol); and preservatives (e.g., methyl or propyl
p-hydroxy benzoates or sorbic acid).
[0140] Granulating techniques are well known in the pharmaceutical
art for modifying starting powders or other particulate materials
of an active ingredient. The powders are typically mixed with a
binder material into larger permanent free-flowing agglomerates or
granules referred to as a "granulation." For example, solvent-using
"wet" granulation processes are generally characterized in that the
powders are combined with a binder material and moistened with
water or an organic solvent under conditions resulting in the
formation of a wet granulated mass from which the solvent must then
be evaporated.
[0141] Melt granulation generally consists in the use of materials
that are solid or semi-solid at room temperature (i.e. having a
relatively low softening or melting point range) to promote
granulation of powdered or other materials, essentially in the
absence of added water or other liquid solvents. The low melting
solids, when heated to a temperature in the melting point range,
liquefy to act as a binder or granulating medium. The liquefied
solid spreads itself over the surface of powdered materials with
which it is contacted, and on cooling, forms a solid granulated
mass in which the initial materials are bound together. The
resulting melt granulation may then be provided to a tablet press
or be encapsulated for preparing the oral dosage form. Melt
granulation improves the dissolution rate and bioavailability of an
active (i.e., drug) by forming a solid dispersion or solid
solution.
[0142] U.S. Pat. No. 5,169,645 discloses directly compressible
wax-containing granules having improved flow properties. The
granules are obtained when waxes are admixed in the melt with
certain flow improving additives, followed by cooling and
granulation of the admixture. In certain embodiments, only the wax
itself melts in the melt combination of the wax(es) and
additives(s), and in other cases both the wax(es) and the
additives(s) will melt.
[0143] The present invention also includes a multi-layer tablet
comprising a layer providing for the delayed release of one or more
compounds of the invention, and a further layer providing for the
immediate release of a medication for treatment of a disease or
disorder. Using a wax/pH-sensitive polymer mix, a gastric insoluble
composition may be obtained in which the active ingredient is
entrapped, ensuring its delayed release.
Parenteral Administration
[0144] For parenteral administration, the compounds may be
formulated for injection or infusion, for example, intravenous,
intramuscular or subcutaneous injection or infusion, or for
administration in a bolus dose and/or continuous infusion.
Solutions, suspensions or emulsions in an oily or aqueous vehicle,
optionally containing other formulatory agents such as suspending,
stabilizing and/or dispersing agents may be used.
Additional Administration Forms
[0145] Additional dosage forms of this invention include dosage
forms as described in U.S. Pat. Nos. 6,340,475, 6,488,962,
6,451,808, 5,972,389, 5,582,837, and 5,007,790. Additional dosage
forms of this invention also include dosage forms as described in
U.S. Patent Applications Nos. 2003/0147952, 2003/0104062,
2003/0104053, 2003/0044466, 2003/0039688, and 2002/0051820.
Additional dosage forms of this invention also include dosage forms
as described in PCT Applications Nos. WO 03/35041, WO 03/35040, WO
03/35029, WO 03/35177, WO 03/35039, WO 02/96404, WO 02/32416, WO
01/97783, WO 01/56544, WO 01/32217, WO 98/55107, WO 98/11879, WO
97/47285, WO 93/18755, and WO 90/11757.
Controlled Release Formulations and Drug Delivery Systems
[0146] In certain embodiments, the formulations of the present
invention may be, but are not limited to, short-term, rapid-offset,
as well as controlled, for example, sustained release, delayed
release and pulsatile release formulations.
[0147] The term sustained release is used in its conventional sense
to refer to a drug formulation that provides for gradual release of
a drug over an extended period of time, and that may, although not
necessarily, result in substantially constant blood levels of a
drug over an extended time period. The period of time may be as
long as a month or more and should be a release which is longer
that the same amount of agent administered in bolus form.
[0148] For sustained release, the compounds may be formulated with
a suitable polymer or hydrophobic material which provides sustained
release properties to the compounds. As such, the compounds for use
the method of the invention may be administered in the form of
microparticles, for example, by injection or in the form of wafers
or discs by implantation. In certain embodiments, the compounds of
the invention are administered to a patient, alone or in
combination with another pharmaceutical agent, using a sustained
release formulation.
[0149] The term delayed release is used herein in its conventional
sense to refer to a drug formulation that provides for an initial
release of the drug after some delay following drug administration
and that mat, although not necessarily, includes a delay of from
about 10 min up to about 12 hours.
[0150] The term pulsatile release is used herein in its
conventional sense to refer to a drug formulation that provides
release of the drug in such a way as to produce pulsed plasma
profiles of the drug after drug administration.
[0151] The term immediate release is used in its conventional sense
to refer to a drug formulation that provides for release of the
drug immediately after drug administration.
[0152] As used herein, short-term refers to any period of time up
to and including about 8 hours, about 7 hours, about 6 hours, about
5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour,
about 40 min, about 20 min, or about 10 min and any or all whole or
partial increments thereof after drug administration after drug
administration.
[0153] As used herein, rapid-offset refers to any period of time up
to and including about 8 hours, about 7 hours, about 6 hours, about
5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour,
about 40 min, about 20 min, or about 10 min, and any and all whole
or partial increments thereof after drug administration.
Dosing
[0154] The therapeutically effective amount or dose of a compound
will depend on the age, sex and weight of the patient, the current
medical condition of the patient and the progression of Parkinson's
Disease in the patient being treated. The skilled artisan will be
able to determine appropriate dosages depending on these and other
factors.
[0155] A suitable dose of a compound of the present invention may
be in the range of from about 0.01 mg to about 5,000 mg per day,
such as from about 0.1 mg to about 1,000 mg, for example, from
about 1 mg to about 500 mg, such as about 5 mg to about 250 mg per
day. The dose may be administered in a single dosage or in multiple
dosages, for example from 1 to 4 or more times per day. When
multiple dosages are used, the amount of each dosage may be the
same or different. For example, a dose of 1 mg per day may be
administered as two 0.5 mg doses, with about a 12-hour interval
between doses.
[0156] It is understood that the amount of compound dosed per day
may be administered, in non-limiting examples, every day, every
other day, every 2 days, every 3 days, every 4 days, or every 5
days. For example, with every other day administration, a 5 mg per
day dose may be initiated on Monday with a first subsequent 5 mg
per day dose administered on Wednesday, a second subsequent 5 mg
per day dose administered on Friday, and so on.
[0157] The compounds for use in the method of the invention may be
formulated in unit dosage form. The term "unit dosage form" refers
to physically discrete units suitable as unitary dosage for
patients undergoing treatment, with each unit containing a
predetermined quantity of active material calculated to produce the
desired therapeutic effect, optionally in association with a
suitable pharmaceutical carrier. The unit dosage form may be for a
single daily dose or one of multiple daily doses (e.g., about 1 to
4 or more times per day). When multiple daily doses are used, the
unit dosage form may be the same or different for each dose.
[0158] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, numerous
equivalents to the specific procedures, embodiments, claims, and
examples described herein. Such equivalents were considered to be
within the scope of this invention and covered by the claims
appended hereto. For example, it should be understood, that
modifications in reaction conditions, including but not limited to
reaction times, reaction size/volume, and experimental reagents,
such as solvents, catalysts, pressures, atmospheric conditions,
e.g., nitrogen atmosphere, and reducing/oxidizing agents, with
art-recognized alternatives and using no more than routine
experimentation, are within the scope of the present
application.
[0159] It is to be understood that wherever values and ranges are
provided herein, all values and ranges encompassed by these values
and ranges, are meant to be encompassed within the scope of the
present invention. Moreover, all values that fall within these
ranges, as well as the upper or lower limits of a range of values,
are also contemplated by the present application.
[0160] The following examples further illustrate aspects of the
present invention. However, they are in no way a limitation of the
teachings or disclosure of the present invention as set forth
herein.
EXAMPLES
[0161] The invention is now described with reference to the
following Examples. These Examples are provided for the purpose of
illustration only, and the invention is not limited to these
Examples, but rather encompasses all variations that are evident as
a result of the teachings provided herein.
Materials and Methods
Cell Isolation and Culture:
[0162] Cultured rat heart microvascular endothelial cells (RHMVEC)
were purchased from VEC Technologies (Rensselaer, N.Y.) and grown
on fibronectin coated plates in MCDB-131 complete medium (VEC
Technologies). BAEC were isolated from bovine aortas obtained from
a local slaughter house and grown in DMEM (high glucose; Cellgro)
supplemented with 10% FBS (Hyclone) and pen/strep. HUVECS were
isolated locally from human umbilical cords and grown in M199
medium (Invitrogen) supplemented with endothelial cell growth
supplement (Invitrogen), 10% FBS, L-glutamine (Invitrogen) and
pen/strep.
T7 PhageLibrary Construction:
[0163] Novagen T7select phage display system was used for the
random screening of peptides that facilitate endothelial cell
uptake in conjunction with a pool of oligonucleotides randomly
coding for 7-mer peptides. The 7-mer random peptide primers
containing HindIII/XhoI sites were designed as follows: Sense
primer: 5'-GCTAGAATTCNNNBNNBNNBNNBNNBNNBNNBAAGCTTACTGCAGTAGCATG-3'
(SEQ ID NO: 9); Anti-sense primer 5'-CATGCTACTGCAGTAAGCTT-3' (SEQ
ID NO: 10); wherein N=A, T, C, G; and B=G, C, T.
[0164] Similar amount of each primer were mixed and annealed at
95.degree. C. for 5 min, then cooled down to room temperature.
Fill-in reactions were then performed by using the Klenow enzyme to
generate blunt ends DNA fragments. After HindIII/XhoI digestion,
0.06 pmol of inserts were ligated into T7select415-lb vector. The
ligation reaction was added directly to T7 packaging Extracts for
in vitro packaging, and a 3.times.10.sup.7 pfu of phage library was
generated. For amplification, the library was inoculated with BL21
culture (OD.sub.600 of 0.5-1.0) and induced with 1 mM IPTG at
37.degree. C. for 2 hours until cell lysis was observed. The lysate
containing phages was clarified by centrifugation at 8000.times.g
for 10 min, the supernatant was titered and aliquots were stored a
4.degree. C.
Phage Selection by Endocytosis in EC and Amplification:
[0165] RHMVEC (80% confluent; approximately 2.times.10.sup.7
cells/100 mm dish) were washed with PBS and pre-incubated in
serum-free medium at 37.degree. C. for 30 min and inoculated with
an extract (5.times.10.sup.9 pfu) of the T7 phage library to reach
a multiplicity of infection (MOI) of 250. After incubation for 1
hour at 37.degree. C., cells were washed with ice-cold PBS and acid
washed with 0.1N HCl, pH 2.2, for 15 seconds to remove unbounded
and weakly associated phages from the cell surface. Cells were then
trypsinized, centrifuged and lysed with sterile deionized water on
ice. Cell debris were removed by centrifugation and the supernatant
containing previously internalized phages were amplified as
described above and titered between each round to ensure that
5.times.10.sup.9 pfu of input phages was used at the start of each
successive round. After completion of six rounds of
selection/amplification, Eshcherichia Coli BL21 was infected with
the resulting phages and plated, individual plaques were picked,
amplified and sequenced.
Peptide Synthesis:
[0166] Peptides, corresponding to Endo5 (RRPPR) (SEQ ID NO: 1) or
Antennapedia (RQIKIWFQNRRMKWKK) (SEQ ID NO: 2) with or without
cargo fused to their C-terminus end (caveolin-1 amino acids 82-101;
DGIWKASFTTFTVTKYWFYR) (SEQ ID NO: 5) were synthesized by standard
Fmoc chemistry and analyzed by mass spectrometry to confirm purity
by the W.M. Keck biotechnology resource center at Yale University
School of Medicine. Fluorophores (carboxyfluorescein for Endo5 and
rhodamine for AP) were added to the N-terminus following
synthesis.
[0167] Before each experiment, desiccated peptides were weighed,
dissolved in dimethyl sulfoxide (DMSO; J. T. Baker, Philipsburg,
N.J.) to 5.times.10.sup.-2-10.sup.-2 M and diluted to 10.sup.-3 M
with distilled water.
NO Release:
[0168] VEGF-induced NO release experiments were performed as
previously described.
[0169] Briefly, confluent BAEC were incubated in serum-free DMEM
for 6 hours with peptides. Media was removed and fresh serum-free
DMEM was added, with or without VEGF (10.sup.-9 M) for 30 min.
Media was collected, cells were trypsinized and counted, and
nitrites levels in the supernatant were determined by using a
Sievers NO chemiluminescence analyzer.
Modified Miles Assay:
[0170] Plasma leakage in mouse skin was studied using the Miles
assay as previously described. Briefly, male swiss mice (30-35 g)
were anesthetized and injected with Evans blue (30 mg/kg in PBS;
Sigma). Phenylisothiocyanate (5% in mineral oil), an analog of
mustard oil (Pierce, Rockford, Ill.) was applied on the right ear
with a cotton tip. The left ear was used as a control and was
treated with mineral oil alone. After 30 min, the anesthetized
animals were sacrificed, perfused, ears were removed, dried and
weighed. Evans blue was extracted from the ears with formamide and
quantified spectrophotometrically at 595 nm.
Quantification of Internalization:
[0171] Cultured BAEC were grown in 6-well plates until confluency
was reached. Cells were washed and incubated in 1 mL of DMEM
containing labeled peptides (10.sup.-6 M) for 1, 2, 4 or 6 hours at
37.degree. C., washed three times with cold PBS containing 0.1 M
glycine (pH 4) to remove non-specific surface staining. After
complete media removal, cells were trypsinized, centrifuged and
proteins were extracted by adding 150 .mu.L of SDS-based or Triton
X-100 lysis buffer. Membranes were removed by centrifugation, and
internalized peptides were quantified by using a fluorescence plate
reader (Perseptive Biosystems). Cells incubated with peptides for 5
min and washed as described were used as basal surface staining.
Linearity of both fluorophores used was determined by performing a
concentration-fluorescence curve using lysis solution. Experiments
with each fluorophore were performed individually to prevent
cross-interference.
CPP Imaging in Live HUVEC:
[0172] Freshly isolated HUVEC were grown in M199 media supplemented
with glutamine, 10% FBS and endothelial cell growth supplement on
Petri dishes with glass bottom. Since CPPs bind non-specifically to
glass, the background fluorescence was reduced by pretreating
glass-bottom Petri dishes with a blocking solution containing
unlabelled AP and EndoS for 30 min (5.times.10.sup.-5 M) in
colorless M199 media with 1% FBS. After cell seeding, media was
removed and carboxyfluorescin-labeled EndoS and Rhodamine-labeled
AP were added to cells (10.sup.-5 M) and cells were incubated at
37.degree. C. and 5% CO.sub.2 for 1 hour (pulse). Media was
removed, cells were rinsed once with warm culture media and peptide
uptake was rapidly visualized on a Zess Axiovert inverted
fluorescence microscope by performing a Z-stack of captured images
followed by volume deconvolution (Openlab software). The new media
was left on cells for an additional 2 hours (total 3 hours) to
chase CPP localization, and cells were visualized again
(chase).
Statistical Analysis:
[0173] Data are mean.+-.S.E. Statistical comparisons were made by
analysis of variance followed by an unpaired Student's t test. Data
were considered significantly different if values of p<0.05 were
observed.
Example 1: Screening of Phage Library for Peptides that Mediate
Phage Internalization
[0174] A T7 phage display library that expresses on average 0.1-1
copy of randomly generated 7-mer peptides on the capsid was
generated. This system was used because the low peptide number on
the capsid makes it suitable for the selection of peptides that
bind strongly to their targets. A constant amount of input phages
(5.times.10.sup.9) was added to cultured RHMVEC and these phages
were selected for their capacity to get quickly internalized
(cellular uptake) by the cell monolayer. After six rounds of
infection/purification, a 100-fold increase in the percentage of
recovered phages was observed, from 0.018 (round 1) to 1.8% (round
6) under identical starting conditions (Table 1), providing
evidence that the resulting phage library displays enhanced
endothelial cell internalization properties. Analysis of the phage
library's capacity of internalization in endothelial cells after
each round of selection suggests an exponential increase in the
uptake percentage (FIG. 1, R.sup.2=0.975 for correlation with
exponential function).
[0175] Following completion of biopanning and enrichment, the
resulting phages were plated, and individual plaques were amplified
and sequenced. Out of the 24 individual phages isolated, five
phages were coding for the unexpectedly short 5-mer peptide RRPPR
(SEQ ID NO: 1), termed EndoS, which was the most frequently
identified peptide (21%). Codon analysis of the DNA sequence of
EndoS coding phage revealed the random and unexpected insertion of
a stop codon in the coding sequence (CGGCGCCCGCCTCGTTGAGGG) (SEQ ID
NO: 8), which rationalized the smaller size of EndoS compared to
the theoretical CPP size our approach can generate (7 amino
acids).
[0176] Without wishing to be limited by any theory, the high
recovery of Endo5 after phage biopanning may be attributable to its
relatively small size compared to theoretical 7-mer peptides for
which the biopanning approach is designed. However, other CPP
ranging from 4 to 7 amino acids were isolated with the technique
described herein, and none of them display the high recovery rate
of Endo5, arguing against a size-dependent selection.
Example 2: eNOS Inhibitory Activity
[0177] AP-Cav blocks agonist-induced eNOS activity in cultured
endothelial cells (Bucci et al., 2000, Nat. Med. 6:1362-7), and
this biological activity is dependent on AP-Cav's internalization,
dosage and pretreatment time. In the present study, the uptake
potential of Endo5 was compared with that of AP by testing the
effect of Endo5 fused to Cav (endo5-Cav) on NO release by cultured
BAEC.
[0178] A six-hour pretreatment of BAEC with AP or Endo5 without
cargo, or with AP-Cav or Endo5-Cav (10.sup.-5 M) had no significant
effect on basal (unstimulated) NO release (FIG. 2A) as assayed by
NO-specific chemiluminescence. Similar pretreatment with AP or
Endo5 showed no significant effect on VEGF-induced NO release,
whereas pretreatment with AP-Cav (10.sup.-5M) blocked VEGF activity
by 48% (Bucci et al., 2000, Nat. Med. 6:1362-7). Interestingly,
similar pretreatment with Endo5-Cav (10.sup.-5 M) completely
impaired VEGF activity on BAEC NO release (FIG. 2A), providing
evidence that Endo5-mediated uptake was more efficient than that of
AP.
[0179] The pharmacological effect of Endo5-Cav on VEGF-induced NO
release was further studied by performing dose-dependent inhibition
experiments. Pretreatment with Endo5-Cav (10.sup.-6-10.sup.-5 M)
caused a dose-dependent inhibition of VEGF-induced NO release (FIG.
2B) with a near-maximum effect at 10.sup.-5 M. AP-Cav activity
reached maximum inhibition at 2.5.times.10.sup.-5M due to peptide
insolubility at greater dose but displayed a much weaker inhibitory
activity (61% inhibition). Analysis of dose response curves
followed by non-linear regression (curve fit) revealed that the
EC.sub.50 of AP-Cav and Endo5-Cav were 1.8.times.10.sup.-6 and
7.5.times.10.sup.-6 M, respectively.
[0180] Since the inhibition of Endo5-Cav on VEGF-induced eNOS
activity was more robust than that of AP-Cav at a similar
concentration, a time-dependent comparison between AP-Cav and
Endo5-Cav effect on eNOS activity was performed. AP-Cav (10.sup.-5
M) had a time-dependent effect on VEGF-induced NO release in BAEC
(FIG. 2C) although a minor difference is observed between 4 h and 6
h incubation time points (57% vs 49%, respectively), suggesting
that a near-complete equilibrium between AP-Cav uptake and
intracellular degradation/elimination may be reached.
[0181] Endo5-Cav inhibitory effect was also time-dependent but more
robust, with a complete eNOS inhibition at 4 hours, suggesting a
faster internalization of Endo5. Moreover, the data indicated that
a six-hour pretreatment with AP-Cav (10.sup.-5 M) had a similar
effect on VEGF-induced NO release as compared to a two-hour
pretreatment with Endo5-Cav at a similar concentration, which
provided evidence that the rate of uptake Endo5-Cav was
approximately three times that of AP-Cav (FIG. 2C).
[0182] The Cav AB domain (amino acids 82-95; SEQ ID NO: 6) mediates
eNOS inhibition (Bernatchez et al., 2005, Proc. Natl. Acad. Sci.
102:761-66). Because Endo5 appears more potent that AP at promoting
cargo internalization, both AP-Cav leader and cargo sequences were
modified in order to maximize the therapeutic effect/size ratio
compared to AP-Cav. Hence, Endo5-CavAB (a 19-mer peptide) was
synthesized and its activity was compared to that of AP-Cav (a
36-mer acid peptide). Pretreatment of BAEC for six hours with
Endo5-CavAB (10.sup.-5 M) completely blocked VEGF-induced NO
release, whereas AP-Cav inhibited VEGF effect by only 52% (FIG. 2D)
at a similar dose. Interestingly, pretreatment with Endo5-CavAB
(2.times.10.sup.-6 M) had a similar effect as AP-Cav (10.sup.-5 M),
inhibiting VEGF-induced NO release by 49%.
[0183] Taken together, these data suggest the feasibility of
optimizing both AP-Cav cell uptake sequence and cargo to maximize
the therapeutic potential per molecule or per amino acid.
Example 3: Anti-Inflammatory Properties
[0184] EC-derived NO production plays an active role in
inflammation, in part by promoting increase in intra-capillary
pressure and subsequent vascular permeability. Pretreatment of mice
with AP-Cav blocks vascular leakage in the Miles assay (Bucci et
al., 2000, Nat. Med. 6:1362-7; Bernatchez et al., 2005, Proc. Natl.
Acad. Sci. USA 102:761-6). This established model may thus be a
valuable tool to asses the in vivo potency of Endo5-fused
peptides.
[0185] An one-hour pretreatment of mice with AP-Cav (1 mg/kg) (FIG.
3A) attenuated by 37% (n=6 per group) compared to mice treated with
AP alone (similar dose on a molecular weight basis). Endo5-Cav
inhibited mustard oil-induced vascular leakage by 58% compared with
Endo5 pretreatment alone (n=6 or 8 per group) and showed a
statistically significant greater inhibitory activity than AP-Cav
<0.05). As illustrated in FIG. 3B, both AP-Cav and Endo5-Cav
attenuate Evans blue extravasation in the ear skin and tissue
compared to control peptides, although Endo5-Cav was more potent.
Without wishing to be limited by any theory, the incomplete
inhibition displayed by Endo5-Cav on mustard oil-induced
inflammation may be explained by the observation that NO plays only
a partial role in mediating vascular permeability in this model
(Bucci et al., 2000, Nat. Med. 6:1362-7).
Example 4: Internalization by Endothelial Cells
[0186] AP is a CPP that crosses the membrane of neurons (Joliot et
al., 1991, Proc. Natl. Acad. Sci. USA 88:1864-8). As discussed
elsewhere herein, Endo5 was unexpectedly found to promote high
internalization of phages in endothelial cells, and Endo5-Cav was
unexpectedly found to be more potent that AP-Cav at preventing eNOS
activation and vascular permeability.
[0187] In the present study, the internalization rate of Endo5 was
directly compared to that of AP by using carboxyfluorescein and
rhodamine-labelled form of each peptide, respectively. The
linearity of each fluorophore-coupled peptide was confirmed by
performing a standard concentration/fluorescence curve. As
illustrated in FIG. 4A, calibration was performed in order to
obtain similar absorbance values for both fluorophores (by
adjusting gain settings for each fluorophores). BAEC were incubated
separately with fluorophore-labelled peptides (10.sup.-6 M) for 1,
2, 4 or 6 hours, acid washed, lysed, and total peptide uptake was
determined by quantifying fluorescence.
[0188] A linear increase in AP internalization with time was
observed (FIG. 4B), peaking at 6 hours at a value of
1.07.times.10.sup.-9 moles of AP/10.sup.6 cells. This indicates
that approximately 11% of the total amount of rhodamine-AP added at
time zero is internalized by a confluent BAEC monolayer in the
settings, providing evidence for an active concentration mechanism.
The rate of internalization of carboxyfluorescein-Endo5 was greater
than that of AP, also peaking at 6 hours with a value of
2.85.times.10.sup.-9 moles of Endo5/10.sup.6 cells, suggesting that
30.5% of the added peptide was internalized after 6 hours (FIG.
4B).
Example 5: Internalization in Live Endothelial Cells
[0189] In prior studies that attempted to shed light on the uptake
mechanisms involved in CPP entry into cells, imaging was performed
in fixed cells. The results of these studies may not be reliable in
view of the mounting evidence that cell fixation leads to the
unexpected nuclear translocation of CPP (Richard et al., 2003, J.
Biol. Chem. 278:585-90).
[0190] In the present study, epifluorescence microscopy experiments
were performed in live HUVEC to compare the uptake mechanism of
Endo5 to AP. After blocking glass bottom Petri dishes with
unlabelled AP and Endo5 to minimize non-specific binding of labeled
peptides, freshly isolated HUVEC were grown to 50% confluent and
labeled with a "pulse" of carboxyfluorescein-Endo5 and rhodamine-AP
(10.sup.-6M) for 1 h, rinsed, followed by a two-hour "chase" for a
total of 3 hours. Deconvoluted images were captured after the
"pulse" and "chase" periods. Both Endo5 (green channel) and AP (red
channel) display diffuse punctate cytoplasmic staining in live
HUVEC after 1 h of incubation at 37.degree. C. (FIG. 5A). Nuclear
staining was nearly completely absent (dark central area).
Interestingly, merged images revealed a high degree of
co-localization between Endo5 and AP, characterized by the yellow
color (FIG. 5A, left). This observation suggests similarity between
Endo5 and AP early internalization pathways. Individual incubation
of HUVEC with rhodamine-AP caused little or no signal in the
carboxyfluorescein-Endo5 channel and vice-versa, suggesting the
absence of significant bleed-through.
[0191] After a two-hour "chase" period in absence of CPP, the
punctate staining for both Endo5 and AP was still noticeable but it
displayed a more concentrated rather than diffuse pattern,
stressing an active intracellular concentration/localization
mechanism (FIG. 5B, left). Merged images again revealed
colocalization between Endo5 and AP during this long-term phase of
intracellular peptide concentration (chase) after initial
internalization from the cell surface. Representative cells were
shown. Taken together, these data illustrated the similarity of the
internalization and intracellular distribution between Endo5 and
AP.
[0192] The similarity of the internalization pathways between Endo5
and AP was confirmed by performing competition studies and
quantifying Endo5 and AP ability to promote cargo entry into cells.
As illustrated in FIG. 5C, AP-Cav (10.sup.-5M) partial effect on
VEGF-induced NO release was blocked by pretreatment with either AP
or Endo5 (5.times.10.sup.-5 M). The near-complete inhibition of
VEGF-induced NO release mediated by Endo5-Cav was partially
prevented by pretreatment with either AP or Endo5
(5.times.10.sup.-5 M). As illustrated in FIG. 5C, the 91%
inhibition of VEGF-induced NO release by Endo5-Cav was prevented by
AP (30% inhibition) or Endo5 (13% inhibition). Taken together,
these results suggest that AP and Endo5 are internalized through
similar pathways in EC.
TABLE-US-00001 TABLE 1 Enrichment of phage internalization capacity
following 6 rounds of biopanning RHMVEC were incubated for 1 hour
with 5.0 .times. 10.sup.9 phages (input), lysed, and recovered
phages were quantified (cell uptake) and amplified for the next
round of biopanning. Recovery percentage is expressed as the ratio
of recovered phages to input phages. Round of Input phages
Recovered phages Recovery infection (supernatant) (cell uptake) % 1
5.0 .times. 10.sup.9 9.4 .times. 10.sup.5 0.018 2 5.0 .times.
10.sup.9 3.8 .times. 10.sup.6 0.076 3 5.0 .times. 10.sup.9 7.5
.times. 10.sup.6 0.15 4 5.0 .times. 10.sup.9 2.8 .times. 10.sup.7
0.56 5 5.0 .times. 10.sup.9 4.5 .times. 10.sup.7 0.90 6 5.0 .times.
10.sup.9 9.0 .times. 10.sup.7 1.8 24 individual phages isolated
[0193] The disclosures of each and every patent, patent
application, and publication cited herein are hereby incorporated
herein by reference in their entirety.
While the invention has been disclosed with reference to specific
embodiments, it is apparent that other embodiments and variations
of this invention may be devised by others skilled in the art
without departing from the true spirit and scope of the invention.
The appended claims are intended to be construed to include all
such embodiments and equivalent variations.
Sequence CWU 1
1
1015PRTArtificial SequenceChemically Synthesized 1Arg Arg Pro Pro
Arg 1 5 216PRTArtificial SequenceChemically Synthesized 2Arg Gln
Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys 1 5 10 15
3178PRTHomo sapiens 3Met Ser Gly Gly Lys Tyr Val Asp Ser Glu Gly
His Leu Tyr Thr Val 1 5 10 15 Pro Ile Arg Glu Gln Gly Asn Ile Tyr
Lys Pro Asn Asn Lys Ala Met 20 25 30 Ala Asp Glu Leu Ser Glu Lys
Gln Val Tyr Asp Ala His Thr Lys Glu 35 40 45 Ile Asp Leu Val Asn
Arg Asp Pro Lys His Leu Asn Asp Asp Val Val 50 55 60 Lys Ile Asp
Phe Glu Asp Val Ile Ala Glu Pro Glu Gly Thr His Ser 65 70 75 80 Phe
Asp Gly Ile Trp Lys Ala Ser Phe Thr Thr Phe Thr Val Thr Lys 85 90
95 Tyr Trp Phe Tyr Arg Leu Leu Ser Ala Leu Phe Gly Ile Pro Met Ala
100 105 110 Leu Ile Trp Gly Ile Tyr Phe Ala Ile Leu Ser Phe Leu His
Ile Trp 115 120 125 Ala Val Val Pro Cys Ile Lys Ser Phe Leu Ile Glu
Ile Gln Cys Ile 130 135 140 Ser Arg Val Tyr Ser Ile Tyr Val His Thr
Val Cys Asp Pro Leu Phe 145 150 155 160 Glu Ala Val Gly Lys Ile Phe
Ser Asn Val Arg Ile Asn Leu Gln Lys 165 170 175 Glu Ile 4151PRTHomo
sapiens 4Met Met Ala Glu Glu His Thr Asp Leu Glu Ala Gln Ile Val
Lys Asp 1 5 10 15 Ile His Cys Lys Glu Ile Asp Leu Val Asn Arg Asp
Pro Lys Asn Ile 20 25 30 Asn Glu Asp Ile Val Lys Val Asp Phe Glu
Asp Val Ile Ala Glu Pro 35 40 45 Val Gly Thr Tyr Ser Phe Asp Gly
Val Trp Lys Val Ser Tyr Thr Thr 50 55 60 Phe Thr Val Ser Lys Tyr
Trp Cys Tyr Arg Leu Leu Ser Thr Leu Leu 65 70 75 80 Gly Val Pro Leu
Ala Leu Leu Trp Gly Phe Leu Phe Ala Cys Ile Ser 85 90 95 Phe Cys
His Ile Trp Ala Val Val Pro Cys Ile Lys Ser Tyr Leu Ile 100 105 110
Glu Ile Gln Cys Ile Ser His Ile Tyr Ser Leu Cys Ile Arg Thr Phe 115
120 125 Cys Asn Pro Leu Phe Ala Ala Leu Gly Gln Val Cys Ser Ser Ile
Lys 130 135 140 Val Val Leu Arg Lys Glu Val 145 150 520PRTHomo
sapiens 5Asp Gly Ile Trp Lys Ala Ser Phe Thr Thr Phe Thr Val Thr
Lys Tyr 1 5 10 15 Trp Phe Tyr Arg 20 614PRTHomo sapiens 6Asp Gly
Ile Trp Lys Ala Ser Phe Thr Thr Phe Thr Val Thr 1 5 10
715DNAArtificial SequenceChemically Synthesized 7cggcgcccgc ctcgt
15821DNAArtificial SequenceChemically Synthesized 8cggcgcccgc
ctcgttgagg g 21952DNAArtificial SequenceChemically
Synthesizedmisc_feature(11)..(11)A, T, C, or
Gmisc_feature(12)..(12)A, T, C, or Gmisc_feature(13)..(13)A, T, C,
or Gmisc_feature(14)..(14)G, C, or Tmisc_feature(15)..(15)A, T, C,
or Gmisc_feature(16)..(16)A, T, C, or Gmisc_feature(17)..(17)G, C,
or Tmisc_feature(18)..(18)A, T, C, or Gmisc_feature(19)..(19)A, T,
C, or Gmisc_feature(20)..(20)G, C, or Tmisc_feature(21)..(21)A, T,
C, or Gmisc_feature(22)..(22)A, T, C, or Gmisc_feature(23)..(23)G,
C, or Tmisc_feature(24)..(24)A, T, C, or Gmisc_feature(25)..(25)A,
T, C, or Gmisc_feature(26)..(26)G, C, or Tmisc_feature(27)..(27)A,
T, C, or Gmisc_feature(28)..(28)A, T, C, or
Gmisc_feature(29)..(29)G, C, or Tmisc_feature(30)..(30)A, T, C, or
Gmisc_feature(31)..(31)A, T, C, or Gmisc_feature(32)..(32)G, C, or
T 9gctagaattc nnnnnnnnnn nnnnnnnnnn nnaagcttac tgcagtagca tg
521020DNAArtificial SequenceChemically Synthesized 10catgctactg
cagtaagctt 20
* * * * *